(19)
Europaisches Patentamt
European Patent Office
Office europeen des brevets
(ii:
EP 1 561 759 A1
(12)
EUROPEAN PATENT APPLICATION
published in accordance with Art. 158(3) EPC
(43) Date of publication:
10.08.2005 Bulletin 2005/32
(21) Application number: 03751456.9
(22) Date of filing: 10.10.2003
(51) int ci 7; C07K 16/18, C12P 21/08,
A61K 39/395, A61 P 35/00,
A61 P 37/02, A61 P 43/00
(86) International application number:
PCT/JP2003/013063
(87) International publication number:
WO 2004/033499 (22.04.2004 Gazette 2004/17)
(84)
Designated Contracting States:
(72)
Inventors:
AT BE BG CH CY CZ DE DK EE ES Fl FR GB GR
OZAKI, Shuji
HU IE IT LI LU MC NL PT RO SE SI SK TR
Tokushima-shi, Tokushima 770-0804 (JP)
Designated Extension States:
ABE, Masahiro
AL LT LV MK
Tokushima-shi, Tokushima 770-0033 (JP)
TSUCHIYA, Masayuki
(30)
Priority: 11.10.2002 JP 2002299289
Gotenba-shi, Shizuoka 412-8513 (JP)
KIMURA, Naoki
(71)
Applicants:
Gotenba-shi, Shizuoka 412-8513 (JP)
CHUGAI SEIYAKU KABUSHIKI KAISHA
KAWAI, Shigeto
Tokyo, 115-8543 (JP)
Gotenba-shi, Shizuoka 412-8513 (JP)
Ozaki, Shuji
Tokushima-shi, Tokushima 770-0804 (JP)
(74)
Representative: VOSSIUS & PARTNER
Abe, Masahiro
Siebertstrasse 4
Tokushima-shi, Tokushima 770-0033 (JP)
81675 Munchen (DE)
(54) CELL DEATH-INDUCING AGENT
(57) To identify antigens of the 2D7 antibody, the
present inventors cloned the 2D7 antigen. The results
suggested that the 2D7 antigen is an HLA class I mole-
cule. Based on this finding, the present inventors exam-
ined whether the 2D7 antibody has cell death-inducing
activity. Nuclei fragmentation was observed when the
2D7 antibody was cross-linked with another antibody,
indicating that cell-death was induced. Further, diabod-
ies of the 2D7 antibody were found to have very strong
cell death-inducing activities, even without the addition
of another antibody. These results indicate that minibod-
ies of an HLA-recognizing antibody can be used as cell
death-inducing agents.
LO
1^-
5
LO
CL
LU
Printed by Jouve, 75001 PARIS (FR)
EP 1 561 759 A1
Description
[0001] The present invention relates to minibodies of antibodies that recognize HLA.
s Background Art
[0002] The HLA class I antigen is formed by a heterodimer of a 45-KD a chain comprising three domains (oil , oc2,
c*3), and a 12-KD |32 microglobulin. The main role of the HLA molecule is to present CD8+T cells with antigenic peptides,
formed from about eight to ten amino acids produced inside cells. As such, it plays a very important role in the immune
10 response and immune tolerance induced by this peptide presentation.
[0003] By ligating HLA class IA antigens with antibodies, cell growth-suppressing and cell death-inducing effects
have been observed in lymphocytes, suggesting that HLA molecules may also be signal transduction molecules.
[0004] More specifically, for example, there are reports showing cell growth suppression of activated lymphocytes
by the B9.12.1 antibody against the oe1 domain of human HLA class IA, the W6/32 antibody against the oe2 domain,
15 and the TP25.99 and A1.4 antibodies against the oe3 domain (non-patent literature 1, 2). Furthermore, two types of
antibodies, MoAb90 and YTH862, against the oil domain have been reported to induce apoptosis in activated lym-
phocytes (non-patent literature 2, 3, 4). Apoptosis induced by these two antibodies has been shown to be a caspase-
mediated reaction (non-patent literature 4), and therefore, HLA class IA antigens expressed in lymphocytes are also
speculated to be involved in apoptosis signal transduction.
20 [0005] Furthermore, the 5H7 antibody against the oc3 domain of human HLA class IA (non-patent literature 5), and
the RE2 antibody against the oc2 domain of mouse HLA class IA (non-patent literature 6) have been also reported to
induce cell death in activated lymphocytes and the like. However, in contrast with the aforementioned apoptosis-in-
ducing antibodies MoAb90 and YTH862, none of the cell deaths induced by these antibodies have been shown to be
mediated by caspase. Accordingly, cell deaths due to 5H7 and RE2 are predicted to be of a type completely different
25 from conventionally known apoptosis mechanisms.
[0006] As described above, there are numerous reports of the cell growth-suppressing actions and cell death-induc-
ing actions of anti-HLA antibodies. However, the antibodies used herein are all in the molecular forms of IgG antibodies,
F(ab')2, or Fab. To date there have been no reports that cell death-inducing activity is enhanced by reducing the
molecular weight of antibodies, as in F(ab')2 and Fab.
30 [0007] The 2D7 antibody is a mouse monoclonal antibody obtained by immunizing Balb/c mice with human myeloma
cells (non-patent literature 7). The 2D7 antibody has been observed to bind very specifically to the cell surface of
various lymphoid tumor cells, however, antigens recognized by the 2D7 antibody have not been identified.
[0008] Prior art literature relating to the present invention of this application is shown below.
35 [Non-patent Document 1] Fayen etal., Int. Immunol. 10: 1347-1358(1998)
[Non-patent Document 2] Genestier et al., Blood 90: 3629-3639 (1 997)
[Non-patent Document 3] Genestier et al., Blood 90: 726-735 (1 997)
[Non-patent Document 4] Genestier et al., J. Biol. Chem. 273: 5060-5066 (1 998)
[Non-patent Document 5] Woodle et al., J. Immunol. 158: 2156-2164 (1997)
40 [Non-patent Document 6] Matsuoka et al., J. Exp. Med. 181: 2007-201 5 (1 995)
[Non-patent Document 7] Goto, etal. Blood 84: 1922 (1994)
Disclosure of the Invention
45 [0009] The primary purpose of this invention is to provide minibodies of antibodies that recognize HLA class IA. A
further objective of this invention is to provide novel therapeutic agents for tumors or autoimmune diseases that utilize
these minibodies.
[0010] To identify antigens of the 2D7 antibody, the present inventors used random hexamers to synthesize cDNAs
from the mRNAs purified from the 2D7 antigen-expressing cells, RPMI8226. These were inserted into the retrovirus
so vector, pMX, and a retroviral expression library was produced. The retrovirus expression library was packaged into a
retrovirus by transfection into BOSC23 cells. 2D7 antigens were screened by infecting NIH3T3 cells with the virus thus
obtained, staining these with 2D7 antibody, and then using FACS to perform expression analysis. Cell lysates were
then prepared from RPMI8226 cells and U266 cells expressing the 2D7 antigen, and 2D7 antigens were identified by
immunoprecipitation. As a result of these examinations, 2D7 antigens were proven to be HLA class I molecules.
55 [001 1] Since the molecules recognized by 2D7 antibodies are HLA class IA, the present inventors examined whether
2D7 antibodies have cell death-inducing activity. More specifically, Jurkat cells were cultured in the presence or absence
of 2D7, with anti-mouse IgG antibody also added. Cell nuclei were stained 48 hours later with Hoechst 33258, and
then checked for cell nuclei fragmentation, which is characteristic of dead cells. As a result, hardly any cell death-
2
EP 1 561 759 A1
inducing activity was observed in Jurkat cells with 2D7 antibody alone; however, by further cross-linking the antibody
with anti-mouse IgG antibody, nuclei fragmentation was observed, a showing confirming that cell death was induced.
[0012] As described, because cross-linking with an anti-mouse IgG antibody is necessary for 2D7 antibody to induce
cell death, it is difficult to clinically apply the 2D7 antibody to tumors or autoimmune diseases. Therefore, the present
s inventors examined the effect of reducing the molecular weight of the 2D7 antibody on cell death induction. More
specifically, genes encoding the variable regions of the 2D7 antibody were cloned from hybridomas. The 2D7 antibody
was then made into diabodies using genetic engineering techniques and the effects on cell death-inducing activity was
examined. Surprisingly, the 2D7 antibody converted to diabodies showed strong cell death-inducing activity within a
very short time and at low doses, even without cross-linking with an anti-mouse IgG antibody. Furthermore, the diabody
10 hardly acted on normal peripheral blood-derived lymphocytes and adherent cells, and specifically induced cell death
in various myeloma cells, T cell leukemia cell lines, and activated lymphocytes. The above-mentioned results show
that the minibodies of antibodies recognizing HLA can be utilized as cell death-inducing agents.
[0013] More specifically, the present invention provides the following [1] to [23]:
15 [1] a minibody that recognizes a human leukocyte antigen (HLA);
[2] the minibody of [1], wherein the HLA is an HLA class I;
[3] the minibody of [2], wherein the HLA class I is an HLA-A;
[4] a minibody derived from a 2D7 antibody;
[5] the minibody of any one of [1 ] to [4], wherein the minibody is a diabody;
20 [6] a minibody of any one of (a) to (d):
(a) a minibody comprising the amino acid sequence of SEQ ID NO: 6;
(b) a minibody functionally equivalent to the minibody of (a), and comprising an amino acid sequence with a
substitution, insertion, deletion and/or addition of one or more amino acids in the amino acid sequence of SEQ
25 ID NO: 6;
(c) a minibody comprising the amino acid sequences of CDRs of SEQ ID NOs: 2 and 4; and
(d) a minibody functionally equivalent to the minibody of (c), and comprising an amino acid sequence with a
substitution, insertion, deletion and/or addition of one or more amino acids in the amino acid sequence of the
CDRs of SEQ ID NOs: 2 and 4;
30
[7] a method for producing an HLA-recognizing antibody having increased activity by converting the HLA-recog-
nizing antibody to a low-molecular-weight antibody;
[8] the method of [7], wherein the HLA is an HLA class I;
[9] the method of [8], wherein the HLA class I is an HLA-A;
35 [10] a method for producing a 2D7 antibody having increased activity by converting the 2D7 antibody to a low-
molecular-weight antibody;
[1 1 ] the method of any one of [7] to [1 0], wherein the conversion step comprises conversion to a diabody;
[12] the method of any one of [7] to [11], wherein the activity is a cell death-inducing activity or a cell growth-
suppressing activity;
40 [13] a cell death-inducing agent, comprising as an active ingredient the minibody of any one of [1] to [6], the
minibody produced by the method of any one of [7] to [12], or a 2D7 antibody;
[14] the cell death-inducing agent of [13] that induces cell death of a B cell or T cell;
[15] the cell death-inducing agent of [14], wherein the B cell or T cell is an activated B cell or activated T cell;
[16] a cell growth-suppressing agent comprising as an active ingredient the minibody of any one of [1] to [6], the
45 minibody produced by the method of any one of [7] to [12], or a 2D7 antibody;
[17] an antitumor agent comprising as an active ingredient the minibody of any one of [1] to [6], the minibody
produced by the method of any one of [7] to [12], or a 2D7 antibody;
[1 8] the antitumor agent of [1 7], wherein the tumor is a blood tumor;
[1 9;] a therapeutic agent for an autoimmune disease, wherein the therapeutic agent comprises as an active ingre-
50 dient the minibody of any one of [1] to [6], the minibody produced by the method of any one of [7] to [12], or a 2D7
antibody;
[20] the cell death-inducing agent of any one of [13] to [15], wherein the antibody is a diabody;
[21 ] the cell growth-suppressing agent of [1 6], wherein the antibody is a diabody;
[22] the antitumor agent of [1 7] or [1 8], wherein the antibody is a diabody; and
55 [23] the therapeutic agent for autoimmune disease of [1 9], wherein the antibody is a diabody;
[0014] The present invention provides minibodies that recognize HLA. The minibodies of this invention are useful
since their activity is elevated. Herein activity refers to a biological action that is caused by binding an antibody to an
3
EP 1 561 759 A1
antigen. Specific examples include cell death-inducing actions, apoptosis-inducing actions, cell growth-suppressing
actions, cell differentiation-suppressing actions, cell division-suppressing actions, cell growth-inducing actions, cell
differentiation-inducing actions, cell division-inducing actions, and cell cycle-regulating actions. Cell death-inducing
actions and cell growth-suppressing actions are preferred.
s [0015] The cells that become the target of the above-mentioned actions, such as cell death-inducing actions and
cell growth-suppressing actions, are not particularly limited, though hemocytes and suspended cells are preferred.
Specific examples of hemocytes include lymphocytes (B cells, T cells), neutrophils, eosinophils, basophils, monocytes
(preferably activated peripheral blood mononuclear cells (PBMC)), and myeloma cells, while lymphocytes (B cells, T
cells), and myeloma cells are preferred, and T cells or B cells (particularly activated B cells or activated T cells) are
10 most preferable. Suspended cells refer to cells that, when cultured, grow in a suspended state without adhering to the
surface of culturing vessels of glass, plastic or the like. On the other hand, adherent cells refer to cells that, when
cultured, adhere to the surface of culturing vessels of glass, plastic or the like.
[0016] In the present invention, administration of the minibodies that recognize HLA can treat or prevent diseases
such as tumors including blood tumors (hematopoietic tumors) (specific examples include leukemia, myelodysplastic
15 syndrome, malignant lymphoma, chronic myelogenic leukemia, plasmacytic disorder (myeloma, multiple myeloma,
macroglobulinemia), and myeloproliferative disease (polycythemia vera, essential thrombocythemia, idiopathic mye-
lofibrosis)), and autoimmune diseases (specific examples include rheumatism, autoimmune hepatitis, autoimmune
thyroiditis, autoimmune bullosis, autoimmune adrenocortical disease, autoimmune hemolytic anemia, autoimmune
thrombycytopenic purpura, autoimmune atrophic gastritis, autoimmune neutropenia, autoimmune orchitis, autoimmune
20 encephalomyelitis, autoimmune receptor disease, autoimmune infertility, Crohn's disease, systemic lupus erythema-
tosus, multiple sclerosis, Basedow's disease, juvenile diabetes, Addison's disease, myasthenia gravis, lens-induced
uveitis, psoriasis, and Behchet's disease).
[0017] In the present invention, HLA refers to human leukocyte antigen. HLA molecules are categorized into class
I and class II. Known examples of class I are HLA-A, B, C, E, F, G, H, J, and such; and known examples of class II are
25 HLA-DR, DQ, DP, and such. The antigens recognized by the antibodies of this invention are not particularly limited,
so long as they are HLA molecules, preferably molecules classified as class I, and more preferably HLA-A.
[0018] In the present invention, a minibody comprises an antibody fragment that lacks a portion of a whole antibody
(for example, whole IgG). The minibodies of the present invention are not particularly limited so long as they can bind
an antigen. There are no particular limitations on the antibody fragments of the present invention, so long as they are
30 portions of a whole antibody, and preferably contain a heavy chain variable region (VH) or a light chain variable region
(VL). More preferably, the antibody fragments contain both a heavy chain variable region (VH) and a light chain variable
region (VL). Specific examples of the antibody fragments include Fab, Fab', F(ab')2, Fv, and scFv (single chain Fv),
but are preferably scFv (Huston, J. S. etal., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883; Plickthun "The Phar-
macology of Monoclonal Antibodies" Vol. 113, Resenburg and Moore Ed., Springer Verlag, New York, pp. 269-315,
35 (1 994)). Such antibody fragments can be prepared by treating an antibody with an enzyme, such as papain or pepsin
for example, to generate antibody fragments, or by constructing genes that encode these antibody fragments, intro-
ducing them into expression vectors, and then expressing them in appropriate host cells (see, for example, Co, M. S.
etal., 1994, J. Immunol. 152,2968-2976; Better, M. and Horwitz, A. H., 1989, Methods Enzymol. 178, 476-496; Pluck-
thun, A. and Skerra, A., 1989, Methods Enzymol. 178, 497-515; Lamoyi, E., 1986, Methods Enzymol. 121, 652-663;
40 Rousseaux, J. etal., 1986, Methods Enzymol. 121, 663-669; Bird, R. E. and Walker, B. W., 1991, Trends Biotechnol.
9, 132-137).
[0019] The minibodies of this invention preferably have smaller molecular weights than a whole antibody, however,
they may form multimers, including dimers, trimers, and tetramers, and the molecular weights may become greater
than that of the whole antibody.
45 [0020] A preferred minibody of this invention is an antibody comprising two or more antibody VHs and two or more
antibody VLs, in which each of these variable regions is linked directly or indirectly via linkers and such. Such linkages
may be covalent bonds or non-covalent bonds, or may be both. An even more preferable minibody is an antibody
comprising two or more VH-VL pairs formed by non-covalent bonding between VH and VL. In this case, the distance
between one VH-VL pair and another VH-VL pair is preferably shorter in a minibody than in a whole antibody.
so [0021] A particularly favorable minibody of this invention is a diabody. A diabody is a dimer formed by bonding two
fragments, in which a variable region is linked to another variable region via a linker and such (for example, scFv)
(hereinafter referred to as diabody-constituting fragments), and usually comprises two VLs and two VHs (P. Holliger
etal., Proc. Natl. Acad. Sci. USA, 90, 6444-6448 (1993); EP404097; W093/1 11 61 ; Johnson etal., Method in Enzy-
mology, 203, 88-98, (1991); Holliger et ai, Protein Engineering, 9, 299-305, (1996); Perisic etal., Structure, 2,
55 1 21 7-1226, (1994); John etal., Protein Engineering, 12(7), 597-604, (1999); Holliger et ai, Proc. Natl. Acad. Sci. USA.,
90, 6444-6448, (1993); Atwell etal., Mol. Immunol. 33, 1301-1312, (1996)). The bonds between the diabody-consti-
tuting fragments may be non-covalent or covalent bonds, but are preferably non-covalent bonds.
[0022] Alternatively, diabody-constituting fragments maybe bound by a linker and such to form a single chain diabody
4
EP 1 561 759 A1
(sc diabody). In such cases, linking the diabody-constituting fragments using a long linker of about 20 amino acids
allows diabody-constituting fragments on the same chain to form a dimer via non-covalent bonds to each other.
[0023] Diabody-constituting fragments include those with a linked VL-VH, linked VL-VL, and linked VH-VH, and are
preferably those with a linked VH-VL. In the diabody-constituting fragments, the linker used to link a variable region to
s a variable region is not particularly limited, but is preferably a linker short enough to prevent non-covalent bonding
between variable regions in the same fragment. The length of such a linker can be appropriately determined by those
skilled in the art, and is ordinarily 2 to 1 4 amino acids, preferably 3 to 9 amino acids, and most preferably 4 to 6 amino
acids. In this case, linkers between a VL and VH encoded on the same fragment are short, and thus a VL and VH on
the same strand do not form a non-covalent bond nor a single-chain V region fragment, rather, the fragment forms a
10 dimer with another fragment via non-covalent bonding. Furthermore, according to the same principle as in diabody
construction, three or more diabody-constituting fragments may be bonded to form multimeric antibodies, such as
trimers and tetramers.
[0024] Examples of the diabodies of this invention are, without limitation, a diabody comprising the amino acid se-
quence of SEQ ID NO: 6, or a diabody that is functionally equivalent to a diabody comprising the sequence of SEQ ID
15 NO: 6, which comprises an amino acid sequence with a mutation (substitution, deletion, insertion, and/or addition) of
one or more amino acids in the amino acid sequence of SEQ ID NO: 6; and a diabody comprising the amino acid
sequence of a complementarity-determining region (CDR) (or a variable region) of SEQ ID NO: 2 and a CDR (or a
variable region) of SEQ ID NO: 4, or a diabody that is functionally equivalent to a diabody comprising the amino acid
sequence of a CDR (or variable region) of SEQ ID NO: 2 and a CDR (or a variable region) of SEQ ID NO: 4, which
20 comprises an amino acid sequence with mutations (substitution, deletion, insertion, and/or addition) of one or more
amino acids in the amino acid sequence of a CDR (or a variable region) of SEQ ID NO: 2 and a CDR (or a variable
region) of SEQ ID NO: 4.
[0025] Herein, "functionally equivalent" means that the diabody of interest has an activity equivalent to an activity of
a diabody comprising the sequence of SEQ ID NO: 6, or a diabody comprising the sequence of a CDR (or a variable
25 region) of SEQ ID NO: 2 and a CDR (or a variable region) of SEQ ID NO: 4 (for example, HLA-A binding activity, and
cell death-inducing activity).
[0026] The number of mutated amino acids is not limited, but may ordinarily be 30 amino acids or less, preferably
15 amino acids or less, and more preferably five amino acids or less (for example, three amino acids or less).
[0027] Furthermore, a diabody comprising the amino acid sequence of SEQ ID NO: 6, or a diabody comprising the
30 sequence of a CDR (or a variable region) of SEQ ID NO: 2 and a CDR (or a variable region) of SEQ ID NO: 4 may be
humanized or chimerized to reduce heterologous antigenicity against humans.
[0028] In the amino acid sequence of SEQ ID NO: 2, amino acids 1 to 134 correspond to the variable region, amino
acids 50 to 54 correspond to CDR1 , amino acids 69 to 85 correspond to CDR2, and amino acids 11 8 to 134 correspond
to CDR3. In the amino acid sequence of SEQ ID NO: 4, amino acids 1 to 128 correspond to the variable region, amino
35 acids 46 to 55 correspond to CDR1 , amino acids 71 to 77 correspond to CDR2, and amino acids 11 0 to 128 correspond
to CDR3.
[0029] In the present invention, the HLA-recognizing minibodies specifically bind to HLA. They are not particularly
limited, so long as they have a biological action. The minibodies of this invention can be prepared by methods well
known to those skilled in the art. For example, as described in the Examples, the antibodies can be prepared based
40 on the sequence of an HLA-recognizing antibody (particularly sequences of the variable regions and sequences of
CDRs), using genetic engineering techniques known to those skilled in the art.
[0030] For the sequence of the HLA-recognizing antibody, a well-known antibody sequence can be used, or an
anti-HLA antibody can be prepared by a method well known to those skilled in the art using HLA as the antigen, and
then the sequence of this antibody can be obtained and then used. Specifically, for example, this can be performed
45 as follows: HLA protein or its fragment is used as a sensitizing antigen to perform immunizations according to conven-
tional immunization methods, the obtained immunocytes are fused with well-known parent cells according to conven-
tional cell fusion methods, and monoclonal antibody-producing cells (hybridomas) are then screened by ordinary
screening methods. Antigens can be prepared by known methods, such as a method using baculoviruses (W098/46777
and such). Hybridomas can be prepared, for example, according to the method of Milstein etal. (Kohler, G and Milstein,
so c., Methods Enzymol. (1981) 73:3-46). When the antigen has low immunogenicity, immunization can be performed
using the antigen bound to immunogenic macromolecules, such as albumin. Thereafter, cDNAs of the variable region
(V region) of the antibody are synthesized from the mRNAs of the hybridomas using reverse transcriptase, and the
sequences of the obtained cDNAs can be determined by known methods.
[0031] Antibodies that recognize HLA are not particularly limited, so long as they bind to HLA; mouse antibodies, rat
55 antibodies, rabbit antibodies, sheep antibodies, human antibodies, and such may be used as necessary. Alternatively,
artificially modified, genetically recombinant antibodies, such as chimeric and humanized antibodies, may be used to
reduce heterologous antigenicity against humans. These modified antibodies can be produced using known methods.
A chimeric antibody is an antibody comprising the variable regions of the heavy and light chains of an antibody from
5
EP 1 561 759 A1
a non-human mammal such as a mouse, and the constant regions of the heavy and light chains of a human antibody.
The chimeric antibody can be produced by linking a DNA encoding the variable regions of the mouse antibody with a
DNA encoding the constant regions of the human antibody, incorporating this into an expression vector, and then
introducing the vector to a host.
s [0032] Humanized antibodies are also referred to as "reshaped human antibodies". Such humanized antibodies are
obtained by transferring the CDR of an antibody derived from a non-human mammal, for example a mouse, to the
CDR of a human antibody, and general gene recombination procedures for this are also known. Specifically, a DNA
sequence designed to link a murine antibody CDR to the framework region (FR) of a human antibody can be synthesized
by PCR, using primers prepared from several oligonucleotides containing overlapping portions of terminal regions.
10 The obtained DNA is linked to a DNA encoding human antibody constant regions, and this is then integrated into an
expression vector, and the antibody is produced by introducing this vector into host cells (see European Patent Appli-
cation EP 239400, and International Patent Application WO 96/02576). The human antibody FR to be linked via the
CDR is selected so the CDR forms a favorable antigen-binding site. To form a suitable antigen-binding site, amino
acids in the framework region of the antibody variable region may be substituted in the CDR of the reshaped human
15 antibody, as necessary (Sato, K. etal., 1993, Cancer Res. 53, 851-856).
[0033] These chimeric antibodies and humanized antibodies can be chimerized, humanized, and such after their
molecular weight is reduced, or their molecular weight can be reduced after they have been chimerized, humanized,
or such.
[0034] Methods for obtaining human antibodies are also known. For example, human lymphocytes can be sensitized
20 in vitro with a desired antigen, or with cells expressing the desired antigen, and the sensitized lymphocytes can be
fused with human myeloma cells, such as U266, to obtain the desired human antibody with antigen-binding activity
(Examined Published Japanese Patent Application No. (JP-B) Hei 1-59878). Further, a desired human antibody can
be obtained by using a desired antigen to immunize transgenic animals that have a full repertoire of human antibody
genes (see International Patent Application WO 93/1 2227, WO 92/0391 8, WO 94/02602, WO 94/25585, WO 96/34096,
25 and WO 96/33735). Furthermore, techniques for obtaining human antibodies by panning using a human antibody library
are also known. For example, variable regions of human antibodies can be expressed as single chain antibodies
(scFvs) on the surface of phages using phage display methods, and phages that bind to antigens can be selected.
The DNA sequences that encode the variable regions of the human antibodies binding the antigens can be determined
by analyzing the genes of the selected phages. By determining the DNA sequences of the scFvs that bind to the
30 antigens, appropriate expression vectors carrying relevant sequences can be produced to yield human antibodies.
These methods are already known, and are detailed in the following publications: WO 92/01047, WO 92/20791, WO
93/06213, WO 93/11236, WO 93/19172, WO 95/01438, and WO 95/15388.
[0035] In the present invention, favorable examples of antibodies that recognize HLA include 2D7 antibodies. Ex-
amples of 2D7 antibodies are antibodies comprising the sequences of a CDR (or a variable region) of SEQ ID NO: 2
35 and a CDR (or a variable region) of SEQ ID NO: 4, but are not limited thereto. The 2D7 antibodies of this invention
include an antibody which is functionally equivalent to an antibody that comprises the sequence of a CDR (or a variable
region) of SEQ ID NO: 2 and a CDR (or a variable region) of SEQ ID NO: 4, and which comprises an amino acid
sequences with a mutation (substitution, deletion, insertion, and/or addition) of one or more amino acids in the amino
acid sequence of a CDR (or a variable region) of SEQ ID NO: 2 and a CDR (or a variable region) of SEQ ID NO: 4.
40 Herein, "functionally equivalent" means that an antibody of interest has an activity (for example, HLA-A binding activity,
and cell death-inducing activity) equivalent to an antibody comprising the sequence of a CDR (or a variable region) of
SEQ ID NO: 2 and a CDR (or a variable region) of SEQ ID NO: 4.
[0036] The number of mutated amino acids is not particularly limited, but may be ordinarily 30 amino acids or less,
preferably 15 amino acids or less, and more preferably five amino acids or less (for example, three amino acids or
45 less). The amino acids are preferably mutated or modified in a way that conserves the properties of the amino acid
side chain. Examples of amino acid side chain properties are: hydrophobic amino acids (A, I, L, M, F, P, W, Y, and V),
hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, and T), amino acids comprising the following side chains: aliphatic
side chains (G, A, V, L, I, and P); hydroxyl-containing side chains (S, T, and Y); sulfur-containing side chains (C and
M); carboxylic acid- and amide-containing side chains (D, N, E, and Q); basic side chains (R, K, and H); aromatic ring-
so containing side chains (H, F, Y, and W) (amino acids are represented by one-letter codes in parentheses). Polypeptides
comprising a modified amino acid sequence, in which one or more amino acid residues is deleted, added, and/or
replaced with other amino acids, are known to retain their original biological activities (Mark, D. F. et al., Proc. Natl.
Acad. Sci. USA 81, 5662-5666 (1984); Zoller, M. J. & Smith, M. Nucleic Acids Research 10, 6487-6500 (1982); Wang,
A. et al., Science 224, 1431-1433; Dalbadie-McFarland, G. etal., Proc. Natl. Acad. Sci. USA 79, 6409-6413 (1982)).
55 In addition, the amino acid sequences of the antibody constant regions and such are well known to those skilled in the
art.
[0037] Furthermore, the 2D7 antibodies can be chimerized, humanized, or such by methods well known to those
skilled in the art. Such chimeric and humanized antibodies are also included in the 2D7 antibodies of this invention.
6
EP 1 561 759 A1
[0038] The antibodies of this invention may be conjugated antibodies that are bonded to various molecules, such as
polyethylene glycol (PEG), radioactive substances, and toxins. Such conjugate antibodies can be obtained by per-
forming chemical modifications on the obtained antibodies. Methods for antibody modification are established in this
field. The term "antibody" in this invention includes such conjugate antibodies.
s [0039] The present invention includes DNAs that encode the antibodies of this invention. This invention also includes
DNAs encoding antibodies that hybridize under stringent conditions to the aforementioned DNAs, and have antigen-
binding capacity and activity. Hybridization techniques (Sambrook, J. etal., Molecular Cloning 2nd ed., 9.47-9.58, Cold
Spring Harbor Lab. press, 1 989) are well known to those skilled in the art, and hybridization conditions can be selected
appropriately by those skilled in the art. Such hybridization conditions include, for example, conditions of low stringency.
10 Examples of conditions of low stringency include post-hybridization washing in 0.1x SSC and 0.1% SDS at 42°C, and
preferably in 0. 1 x SSC and 0. 1 % SDS at 50°C. More preferable hybridization conditions include those of high stringency.
Highly stringent conditions include, for example, washing in 5x SSC and 0.1% SDS at 65°C. In these conditions, the
higher the temperature, the higher the expectation of efficiently obtaining DNAs with a high homology. However, several
factors, such as temperature and salt concentration, can influence hybridization stringency, and those skilled in the art
15 can suitably select these factors to achieve similar stringencies.
[0040] The DNAs of this invention are used for in vivo and in vitro production of the antibodies of this invention, and
for other applications, such as gene therapy. The DNAs of this invention may be in any form, so long as they encode
the antibodies of this invention. More specifically, they may be cDNAs synthesized from mRNAs, genomic DNAs,
chemically synthesized DNAs, or such. Furthermore, the DNAs of this invention include any nucleotide sequence based
20 on the degeneracy of the genetic code, so long as they encode the antibodies of this invention.
[0041] The antibodies of this invention can be produced by methods well known to those skilled in the art. More
specifically, a DNA of an antibody of interest is incorporated into an expression vector. In so doing, the DNA is incor-
porated into the expression vector and expressed under the control of an expression regulatory region such as an
enhancer or promoter. Next, antibodies can be expressed by transforming host cells with this expression vector. In this
25 regard, appropriate combinations of hosts and expression vectors can be used.
[0042] The vectors include, for example, M1 3 vectors, pUC vectors, pBR322, pBluescript, and pCR-Script. In addition
to the above vectors, for example, pGEM-T, pDIRECT, and pT7 can also be used for the subcloning and excision of
cDNAs.
[0043] When using vectors to produce the antibodies of this invention, expression vectors are particularly useful.
30 When an expression vector is expressed in E. coli, for example, it should have the above characteristics in order to
be amplified in E. coli. Additionally, when E. coli such as JM109, DH5 oc , HB101, or XL1-Blue are used as the host
cell, the vector preferably has a promoter, for example, a lacZ promoter (Ward etal. (1 989) Nature 341 :544-546; (1 992)
FASEB J. 6:2422-2427), araB promoter (Better et al. (1988) Science 240:1041-1043), orT7 promoter, to allow efficient
expression of the desired gene in E. coli. Other examples of the vectors include pGEX-5X-1 (Pharmacia), "QIAexpress
35 system" (QIAGEN), pEGFP, and pET (where BL21 , a strain expressing T7 RNA polymerase, is preferably used as the
host).
[0044] Furthermore, the vector may comprise a signal sequence for polypeptide secretion. When producing proteins
into the periplasm of E. coli, the pelB signal sequence (Lei, S. P. et al. J. Bacterid. 169:4379 (1987)) may be used as
a signal sequence for protein secretion. For example, calcium chloride methods or electroporation methods may be
40 used to introduce the vector into a host cell.
[0045] In addition to E. coli, expression vectors derived from mammals (e.g., pCDNA3 (Invitrogen), pEGF-BOS (Nu-
cleic Acids Res. (1990) 1 8(1 7):5322), pEF, pCDM8), insect cells (e.g., "Bac-to-BAC baculovirus expression system"
(GIBCO-BRL), pBacPAK8), plants (e.g., pMH1, pMH2), animal viruses (e.g., pHSV, pMV, pAdexLcw), retroviruses (e.
g., pZIPneo), yeasts (e.g., "Pichia Expression Kit" (Invitrogen), pNV11, SP-Q01), and Bacillus subtilis (e.g., pPL608,
45 pKTH50) may also be used as a vector for producing the polypeptide of the present invention.
[0046] In order to express proteins in animal cells, such as CHO, COS, and NIH3T3 cells, the vector preferably has
a promoter necessary for expression in such cells, for example, an SV40 promoter (Mulligan et al. (1 979) Nature 277:
108), MMLV-LTR promoter, EFIocpromoter (Mizushima ef a/. (1990) Nucleic Acids Res. 18:5322), CMV promoter, etc.).
It is even more preferable that the vector also carry a marker gene for selecting transformants (for example, a drug-
so resistance gene enabling selection by a drug, such as neomycin and G418). Examples of vectors with such charac-
teristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP 13, and such.
[0047] In addition, to stably express a gene and amplify the gene copy number in cells, CHO cells having a defective
nucleic acid synthesis pathway can be introduced with a vector containing a DHFR gene (for example, pCHOI) to
compensate for the defect, and the copy number may be amplified using methotrexate (MTX). Alternatively, a COS
55 cell, which carries an SV40 T antigen-expressing gene on its chromosome, can be transformed with a vector containing
the SV40 replication origin (for example, pcD) for transient gene expression. The replication origin may be derived
from polyoma viruses, adenoviruses, bovine papilloma viruses (BPV), and such. Furthermore, to increase the gene
copy number in host cells, the expression vector may contain, as a selection marker, an aminoglycoside transferase
7
EP 1 561 759 A1
(APH) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene, dihy-
drofolate reductase (dhfr) gene, and such.
[0048] Methods for expressing the DNAs of this invention in the bodies of animals include methods of incorporating
the DNAs of this invention into appropriate vectors and introducing them into living bodies by, for example, a retrovirus
s method, liposome method, cationic liposome method, or adenovirus method. The vectors that are used include ade-
novirus vectors (for example, pAdexIcw), and retrovirus vectors (for example, pZIPneo), but are not limited thereto.
General genetic manipulations such inserting the DNAs of this invention into vectors can be performed according to
conventional methods (Molecular Cloning, 5.61-5.63). Administration to living bodies can be carried out by ex vivo
method or in vivo methods.
w [0049] Furthermore, the present invention provides host cells into which a vector of this invention is introduced. The
host cells into which a vector of this invention is introduced are not particularly limited; for example, E. coli and various
animal cells are available for this purpose. The host cells of this invention may be used, for example, as production
systems to produce and express the antibodies of the present invention. In vitro and in vivo production systems are
available for polypeptide production systems. Production systems that use eukaryotic cells or prokaryotic cells are
15 examples of in vitro production systems.
[0050] Eukaryotic cells that can be used include, for example, animal cells, plant cells, and fungal cells. Known animal
cells include: mammalian cells, for example, CHO (J. Exp. Med. (1995)108, 945), COS, 3T3, myeloma, BHK (baby
hamster kidney), HeLa, Vero, amphibian cells such as Xenopus laevis oocytes (Valle, ef al. (1981) Nature 291,
358-340), or insect cells (e.g., Sf9, Sf21, and Tn5). CHO cells in which the DHFR gene has been deleted, such as
20 dhfr-CHO (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220) and CHO K-1 (Proc. Natl. Acad. Sci. USA (1968) 60,
1275), are particularly preferable for use as CHO cells. Of the animal cells, CHO cells are particularly favorable for
large-scale expression. Vectors can be introduced into a host cell by, for example, calcium phosphate methods, DEAE-
dextran methods, methods using cationic liposome DOTAP (Boehringer-Mannheim), electroporation methods, lipofec-
tion methods, etc.
25 [0051] Plant cells include, for example, Nicotiana tabacum-derived cells known as polypeptide production systems.
Calluses may be cultured from these cells. Known fungal cells include yeast cells, for example, the genus Saccharo-
myces, such as Saccharomyces cerevisiae; and filamentous fungi, for example, the genus Aspergillus such as As-
pergillus niger.
[0052] Bacterial cells can be used in prokaryotic production systems. Examples of bacterial cells include E. coli (for
30 example, JM109, DH5oc, HB101 and such); and Bacillus subtilis.
[0053] Antibodies can be obtained by transforming the cells with a polynucleotide of interest, then culturing these
transformants in vitro. Transformants can be cultured using known methods. For example, DMEM, MEM, RPMI 1640,
or IMDM may be used as the culture medium for animal cells, and may be used with or without serum supplements
such as fetal calf serum (FCS). Serum-free cultures are also acceptable. The preferred pH is about 6 to 8 over the
35 course of culturing. Incubation is typically carried out at a temperature of about 30 to 40°C for about 15 to 200 hours.
Medium is exchanged, aerated, or agitated, as necessary.
[0054] On the other hand, production systems using animal or plant hosts may be used as systems for producing
polypeptides in vivo. For example, a DNA of interest may be introduced into an animal or plant, and the polypeptide
produced in the body of the animal or plant is then recovered. The "hosts" of the present invention include such animals
40 and plants.
[0055] When using animals, there are production systems using mammals or insects. Mammals such as goats, pigs,
sheep, mice, and cattle may be used (Vicki Glaser SPECTRUM Biotechnology Applications (1993)). Alternatively, the
mammals may be transgenic animals.
[0056] For example, a DNA of interest may be prepared as a fusion gene with a gene encoding a polypeptide spe-
45 cifically produced in milk, such as the goat fi-casein gene. DNA fragments containing the fusion gene are injected into
goat embryos, which are then introduced back to female goats. The desired antibody can then be obtained from milk
produced by the transgenic goats, which are born from the goats that received the embryos, or from their offspring.
Appropriate hormones may be administered to increase the volume of milk containing the polypeptide produced by
the transgenic goats (Ebert, K.M. et al., Bio/Technology 12, 699-702 (1994)).
so [0057] Insects, such as silkworms, may also be used. Baculoviruses carrying a DNA of interest can be used to infect
silkworms, and the antibody of interest can be obtained from their body fluids (Susumu, M. et al., Nature 315, 592-594
(1985)).
[0058] When using plants, tobacco can be used, for example. When tobacco is used, a DNA of interest may be
inserted into a plant expression vector, for example, pMON 530, and then the vector may be introduced into a bacterium,
55 such as Agrobacterium tumefaciens. The bacteria are then used to infect tobacco, such as Nicotiana tabacum, and
the desired polypeptides are recovered from the leaves (Julian K.-C. Ma et al., Eur. J. Immunol. 24, 131-138 (1994)).
[0059] The resulting antibodies of this invention may be isolated from the inside or outside (such as the medium) of
host cells, and purified as substantially pure and homogenous antibodies. Any standard method for isolating and pu-
8
EP 1 561 759 A1
rifying antibodies may be used, and methods are not limited to any specific method. Antibodies may be isolated and
purified by selecting an appropriate combination of, for example, chromatographic columns, filtration, ultrafiltration,
salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electro-
phoresis, isoelectric focusing, dialysis, recrystallization, and others.
s [0060] Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic
chromatography, gel filtration, reverse-phase chromatography, and adsorption chromatography (Strategies for Protein
Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak etal., Cold Spring Harbor Lab-
oratory Press, 1 996). These chromatographies can be carried out using liquid phase chromatographies such as HPLC
and FPLC. The present invention also includes antibodies that are highly purified using these purification methods.
10 [0061] In the present invention, the antigen-binding activity of antibodies (Antibodies A Laboratory Manual. Ed Har-
low, David Lane, Cold Spring Harbor Laboratory, 1988) can be measured using well known techniques. For example,
ELISA (enzyme linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), or fluoroimmu-
noassay may be used.
[0062] In the present invention, whether or not the antibodies of this invention induce cell death in suspended cells
15 can be determined from whether cell death is induced in Jurkat cells or ARH77 cells, as in the Examples. Whether or
not the antibodies induce cell death in adhesion cells can be determined from whether cell death is induced in HeLa
cells, as in the Examples.
[0063] Furthermore, the present invention provides cell death-inducing agents or cell growth-suppressing agents
which comprise minibodies or 2D7 antibodies of this invention as active ingredients. The cell death-inducing activity
20 of the minibodies or 2D7 antibodies in this invention is considered to have a particularly large effect on activated T
cells or B cells, therefore, it is considered to be particularly effective for treatment and prevention of tumors such as
cancer (particularly blood tumors), and autoimmune diseases. Accordingly, the present invention provides methods of
treatment and prevention of tumors such as cancer (particularly blood tumors), and autoimmune diseases that use the
minibodies or 2D7 antibodies of this invention. When using 2D7 antibodies whose molecular weight has not been
25 reduced as active ingredients, they are preferably cross-linked with an anti-IgG antibody and such.
[0064] The above-mentioned antibodies can also be used as conjugate antibodies, after linking to various reagents.
Examples of such reagents include chemotherapy reagents, radioactive substances, and toxins. Such conjugate an-
tibodies can be produced by known methods (US5057313, and US5156840).
[0065] The above-mentioned pharmaceutical agents can be directly administered to patients, or administered as
30 pharmaceutical compositions formulated by known pharmaceutical methods. For example, they may be administered
orally, as tablets, capsules, elixirs, or microcapsules, sugar-coated as necessary; or parenterally, in the form of injec-
tions of sterile solution or suspensions prepared with water or other pharmaceutical^ acceptable liquids. For example,
they may be formulated by appropriately combining them with pharmaceutical^ acceptable carriers or media, more
specifically, sterilized water or physiological saline solutions, vegetable oils, emulsifiers, suspending agents, sur-
35 factants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binding agents, and such, and mixing them
at a unit dosage form required for generally accepted pharmaceutical practice. The amount of active ingredient in the
formulation is such that appropriate doses within indicated ranges are achieved.
[0066] Additives that can be mixed into tablets and capsules include, for example, binding agents such as gelatin,
cornstarch, tragacanth gum, and gum arabic; excipients such as crystalline cellulose; swelling agents such as corn-
40 starch, gelatin, alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, or saccha-
rine; and flavoring agents such as peppermint and Gaultheria adenothrix oils, or cherry. When the unit dosage form is
a capsule, liquid carriers, such as oils and fats, can be further included in the above-indicated materials. Sterile com-
positions to be injected can be formulated using a vehicle such as distilled water used for injection, according to standard
protocols.
45 [0067] Aqueous solutions used for injections include, for example, physiological saline and isotonic solutions com-
prising glucose or other adjunctive agents such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. They may
also be combined with appropriate solubilizing agents, such as alcohol, and specifically, ethanol, polyalcohol such as
propylene glycol or polyethylene glycol, or non-ionic detergent such as polysorbate 80™ or HCO-50, as necessary.
[0068] Oil solutions include sesame oils and soybean oils, and can be combined with solubilizing agents such as
so benzyl benzoate or benzyl alcohol. Injection solutions may also be formulated with buffers, for example, phosphate
buffers or sodium acetate buffers; analgesics, for example, procaine hydrochloride; stabilizers, for example, benzyl
alcohol or phenol; or anti-oxidants. The prepared injections are typically aliquoted into appropriate ampules.
[0069] Administration to patients may be performed, for example by intra-arterial injection, intravenous injection, or
subcutaneous injection, alternatively by intranasal, transbronchial, intramuscular, transdermal, or oral administration
55 using methods well known to those skilled in the art. Doses vary depending on the body weight and age of the patient,
method of administration and such; nevertheless, those skilled in the art can appropriately select suitable doses. Fur-
thermore, if a compound can be encoded by a DNA, the DNA may be incorporated into a gene therapy vector to carry
out gene therapy. Doses and administration methods vary depending on the body weight, age, and symptoms of pa-
9
EP 1 561 759 A1
tients, but, again, they can be appropriately selected by those skilled in the art.
[0070] A single dose of a pharmaceutical agent of this invention varies depending on the target of administration,
the target organ, symptoms, and administration method. However, an ordinary adult dose (presuming a body weight
of 60 kg) in the form of an injection is approximately 0.1 to 1 000 mg, preferably approximately 1 .0 to 50 mg, and more
s preferably approximately 1 .0 to 20 mg per day, for example.
[0071] When administered parenterally, a single dose varies depending on the target of administration, the target
organ, symptoms, and administration method, but in the form of an injection, for example, a single dose of approximately
0.01 to 30 mg, preferably approximately 0.1 to 20 mg, and more preferably approximately 0.1 to 10 mg per day may
be advantageously administered intravenously to an ordinary adult (presuming a body weight of 60 kg). For other
10 animals, a converted amount based on the amount for a body weight of 60 kg, or a converted amount based on the
amount for a body surface area can be administered.
Brief Description of the Drawings
15 [0072]
Fig. 1 shows the adaptors used to produce the pMX2 vector. The bold letters indicate BstXI recognition sequences.
Fig. 2A and Fig. 2B show 2D7 antigen expression in cell lines. Each cell type was stained with 2D7 antibody and
their expressions were examined. (Solid line: no primary antibody; dotted line: 2D7 antibody)
20 Fig. 3 is a set of photographs showing the results of immunoprecipitation using the 2D7 antibody. NIH3T3,
RPMI8226, and U266 cells were solubilized, immunoprecipitation was performed with the 2D7 antibody, anti-BST-
1 antibody (control), or protein G itself, and the proteins were detected by silver staining. In RPMI8226 and U266,
a molecule of approximately 1 2 KD (arrow), which is specifically precipitated by the 2D7 antibody, is detected. This
band was cut out and peptide sequenced, and thus found to be |32-microglobulin.
25 Fig. 4 shows flow diagrams for screening. Separation into pools, preparation of DNA, packaging into virus, infection
of 3T3 cells, and screening using FACS were performed in one span (Fig. 4A). By the end of the fourth screening,
the library was narrowed down to approximately 20 clones. In the fifth screening, 64 colonies were individually
inoculated into a 96-well plate, pools were formed using the vertical and horizontal rows, and then screened. As
a result, the library was narrowed down to twelve candidate clones (Fig. 4B).
30 Fig. 5 shows the results of screening using FACS. Fig. 5A shows the results of the second screening, Fig. 5B
shows the results of the third screening, and Fig. 5C shows the results of the fourth screening. NIH3T3 cells were
infected with retroviruses prepared from each pool, and three days later the cells were stained with the 2D7 anti-
body. The clones were narrowed down by gradually reducing the pool size of each screening.
Fig. 6 shows the results of screening using FACS. Fig. 6A shows the results of the fifth screening, and Fig. 6B
35 shows the result of the final screening. As a result of the fifth screening, positive clones were found in rows 3, 4,
6, and 8, and in rows E, F, and G As a result of screening the twelve candidate clones, positive clones were found
in row E at 6E . When the nucleotide sequence of this 6E was analyzed, it was found to encode HLA classl A*6802.
Fig. 7 is a graph and a set of photographs showing the influence on cells of the addition of 2D7 antibody. 2D7
antibody (1 0 ng/ml) was added, and the number of viable cells was determined 48 hours later. Hardly any change
40 in cell growth was observed, even after 2D7 antibody was added (Fig. 7A). K562 cells (Fig. 7B), Jurkat cells (Fig.
7C), and RPMI8226 cells (Fig. 7D) were each observed 24 hours after antibody addition. The 2D7 antibody induced
aggregation of Jurkat cells.
Fig. 8 is a set of photographs showing cell death induction due to cross-linking of the 2D7 antibody. Each combi-
nation of the 2D7 antibody with anti-mouse IgG was made to act on Jurkat cells, and the cell nuclei were stained
45 48 hours later. Nuclear fragmentation due to cell death was observed when the 2D7 antibody and anti-mouse IgG
acted on cells simultaneously.
Fig. 9 shows a 2D7 diabody (2D7DB) sequence.
Fig. 1 0A and Fig. 1 0B show a 2D7 diabody structure. Fig. 1 0C is a photograph showing its transient expression
in COS7 cells.
so Fig. 1 1 A and Fig. 1 1 B show the cytotoxic activity of 2D7DB transiently expressed in COS7.
Fig. 12 shows the cytotoxic activity of 2D7DB transiently expressed in COS7. K562 cells (Fig. 12A) and Jurkat
cells (Fig. 12B) were used.
Fig. 13 shows the cytotoxic activity of 2D7DB transiently expressed in COS7. RPMI8226 cells (Fig. 13A), IL-KM3
cells (Fig. 13B), U266 cells (Fig. 13C), and ARH77 cells (Fig. 13D) were used.
55 Fig. 14 is a graph showing the growth-suppressing effect of purified 2D7DB.
Fig. 15 shows cell death induction by purified 2D7DB, 48 hours after induction. ARH77 cells (Fig. 15A), Jurkat
cells (Fig. 15B), K562 cells (Fig. 15C), and HeLa cells (Fig. 15D) were used.
Fig. 1 6 shows cell death induction by purified 2D7DB, 48 hours after induction. U266 cells (Fig. 1 6A), and IL-KM3
10
EP 1 561 759 A1
cells (Fig. 1 6B) were used for the study.
Fig. 1 7 shows a time course of cell death induction by 2D7DB (2 ng/ml). Cell death induction was investigated at
12 through to 38 hours. ARH77 cells (Fig. 17A) and Jurkat cells (Fig. 17B) were used.
Fig. 1 8 shows a time course of cell death induction by 2D7DB (2 ng/ml). Cell death induction was investigated at
s three through to six hours. ARH77 cells (Fig. 1 8A) and Jurkat cells (Fig. 1 8B) were used.
Fig. 1 9 shows the effect of Z-VAD-FMK on cell death due to 2D7DB. The study was performed using ARH77 cells
16 hours after induction.
Fig. 20 shows the effect of Z-VAD-FMK on cell death due to 2D7DB. The study was performed using Jurkat cells
16 hours after induction.
10 Fig. 21 is a set of photographs showing that cell death due to 2D7DB is not accompanied by DNA fragmentation.
The study was performed 24 hours after cell death induction.
Fig. 22 shows the results of investigating the effect of cytochalasin D on the cell death-inducing activity of 2D7DB.
By pre-treating ARH77 cells with cytochalasin D, which is an actin-polymerization inhibitor, the cells showed re-
sistance to 2D7DB-induced cell death.
15 Fig. 23 is a set of photographs showing the results of immunostaining to investigate the state of the intracellular
actin and nuclei. After reacting ARH77 cells under the conditions described in the figure, actin was detected using
anti-actin antibody (red), and cell nuclei were detected using Hoechst 33258 (blue). Actin was absent from cells
treated with 2D7DB.
Fig. 24 shows that the 2D7DB suppresses an increase in human IgG (hlgG) concentration in serum in a mouse
20 model of human myeloma. The data shows the average + SEM. There was a significant difference (*: p<0.05)
between the vehicle-administered group and the 2D7DB-administered group, according to unpaired t-tests.
Fig. 25 shows that the 2D7DB has a life-prolonging effect in a mouse model of human myeloma. There was a
significant difference (*: p<0.05) between the vehicle-administered group and the 2D7DB-administered group,
according to generalized Wilcoxon tests.
25 Fig. 26 shows analyses of the action of 2D7DB on PBMC. PHA-M (Fig. 26A), ConA (Fig. 26B), and SAC (Fig. 26C)
were used as mitogens. Fig. 26D shows the results in the absence of a mitogen, and Fig. 26E shows the results
of a positive control (ARH77). The results shown are, from the top, those of no 2D7DB addition, three-hour addition,
and 24-hour addition.
30 Best Mode for Carrying out the Invention
[0073] Herein below, the present invention is specifically described using Examples; however, it should not to be
construed as being limited thereto.
35 [1] Cell lines
[0074] Human myeloma cell lines (RPMI8226, K562, and ARH77), human T-cell leukemiacell line (Jurkat), FDC-P1 ,
HCI-16, and 2D7 hybridomacell line (from University of Tokushima) were cultured in RPMI1640 medium (GIBCO BRL)
supplemented with 10% fetal calf serum (FCS). Human myeloma cell lines (IL-KM3 and U266) were individually cultured
40 in the same medium supplemented with 2 ng/ml of IL-6 (R & D), and Ba/F3 was cultured in the same medium supple-
mented with 2 ng/ml of IL-3 (R & D). COS7, 293T, HeLa, NIH3T3, and BOSC23 were cultured in DMEM medium
(GIBCO BRL) supplemented with 10% FCS, and CHO was cultured in cc-MEM medium (GIBCO BRL) supplemented
with 5% FCS or 10% FCS.
45 [2] Production of pMX2 vectors
[0075] The GFP gene region of the retrovirus vector, pMX-GFP, which packages the GFP gene in the virus particle,
was cut out and removed using EcoRI-Sall. The adaptor, which comprised a BstXI site in its sequence (Fig. 1) (and
was synthesized with an ABI DNA synthesizer, then annealed in vitro before use), was inserted into this region, forming
so pMX2.
[3] Production of cDNA libraries
[0076] Total RNA was purified from RPMI8226 cells by standard methods using Trisol (GIBCO BRL). Furthermore,
55 the mRNAs were purified from 200 |xg of this total RNA, using a uMACS mRNA Isolation kit (Miltenyi Biotec) according
to the manufacturer's instructions. The cDNAs were synthesized using random hexamers (Superscript Choice System
for cDNA Synthesis; Invitrogen) with 3.6 [ig of mRNA as template, and then a BstXI adaptor (Invitrogen) was linked to
both ends. This cDNA was inserted into a pMX2 vector cleaved with BstXI, and was introduced into ELECTRO MAX
11
EP 1 561 759 A1
DH10B (GIBCO BRL) by electroporation (2.5 KV, 200 n, 25 |^F). After adding 1 ml of SOC, the vectors were then
incubated at 37°C for one hour, 1 ml of 40% glycerol/LB+Amp was added. A portion of the culture was used to check
the titer and the remainder was stored at -80°C. The obtained library was plated at 200 ^l/well (7% DMSO/LB+Amp)
into two 96-well plates, so that each well contained 1 000 clones. These were cultured overnight at 37°C. Four wells
s (4000 clones) from this plate were combined and placed into an ampicillin-containing LB medium (4 ml). This was
defined as one pool, the rest of the wells were treated similarly. Ultimately, 24 pools were prepared from a single plate.
After incubating each pool overnight at 37°C, DNAs were prepared (QIAGEN) and used for transfection into packaging
cells. The plates used for inoculation were stored at -80°C until used for secondary screening.
10 [4] Purification of antibodies
[0077] 0.5 ml of ascites, sent from University of Tokushima, was adsorbed to a Protein A Hi Trap Affinity column
(Amersham Pharmacia). The IgG fraction was then eluted using 0.1 M sodium citrate, pH3.0, and the 2D7 antibody
was collected. This was concentrated using Centricon (YM-10; Millipore), and the buffer was exchanged to PBS to
15 ultimately yield a total of 5.34 mg of antibody. This was separated into aliquots and stored at -20°C (concentration:
0.89 ng/|xL).
[5] FACS
20 [0078] Adherent cells were detached using 1 mM EDTA/PBS, and suspended cells were collected by centrifugation,
then suspended in FACS buffer (2.5% FCS, 0.02% NaN 3 /PBS). These cells were left to stand on ice for one hour in a
buffer (5% FCS/PBS) containing 2D7 antibody (final concentration 10 ng/ml). These were then washed with FACS
buffer, reacted in a solution of FITC-anti-mouse IgG (Immunotech) (1 :150, 50 \A. FACS buffer) on ice for 30 minutes,
washed twice with FACS buffer, and then analyzed using EPICS ELITE (COULTER).
25
[6] Retrovirus infection
(i) Retrovirus packaging
30 [0079] The day before transfection, 2ml of BOSC23 cells, which are retrovirus-packaging cells, were plated onto a
6-well plate at 6 x 10 5 cells/well. Transfection was carried out by the following procedure: 1 of the plasmid DNA
derived from each pool was mixed with 3 |u.L of FuGENE 6 Transfection Reagent (Roche), left to stand at room tem-
perature for 20 minutes, and then added to the BOSC23 cell culture medium plated the day before. Cells were then
cultured at 37°C for 48 hours, and the culture medium was collected. Dead cells were removed by centrifugation at
35 3000 rpm for five minutes, and the culture solution was then used as the virus solution.
(ii) Virus infection
[0080] The 2 ml ofNIH3T3 cells plated onto 6-well plates at 1 x 10 5 cells/well the day before were cultured for 24
40 hours in 1 ml of virus solution supplemented with 10 jig/ml of polybrene (hexadimethrine bromide; Sigma). 1.5 ml of
fresh medium was then added, the cells were cultured for another 48 hours, and gene expression was then analyzed
using FACS.
[7] Immunoprecipitation
45
[0081] Cells were lysed in a lysis buffer (0.5% Nonidet P-40, 10 mM Tris, pH 7.6, 150 mM NaCI, 5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 5 j^g/ml aprotinin), and the resulting solution was centrifuged to remove the insoluble
proteins and obtain a cell lysate. 1 [ig of 2D7 antibody was added, and incubated at 4°C for four hours. Magnetic protein
G (BioMag) was then added, and this was incubated for another one hour. Subsequently, the immunoconjugate was
so washed three times with a lysis buffer, and then subjected to SDS-PAGE. This gel was silver stained (Daiichi Pure
Chemicals) according to the attached instructions. On the other hand, for peptide sequencing, the gel on which
SDS-PAGE was performed was transferred to ProBlott (Applied Biosystems), and this was stained for one minute with
Coomassie blue staining solution (0.1 % coomassie blue R-250 in 40% MetOHV 1% acetic acid). After washing several
times with 50% MetOH, the band of interest was cut out, washed five times with 1 ml of DDW, dried in vacuo, and then
55 subjected to peptide sequencing.
12
EP 1 561 759 A1
[8] Cell growth assay using the 2D7 antibody
[0082] Each type of cell was plated into a 96-well plate at 1 x 10 6 cells/ml in the presence or absence of PMA (50
ng/ml; GIBCO BRL) and PHA (1 0 nl/ml; GIBCO BRL). After subsequent addition (1 0 jig/ml) or no addition of the 2D7
s antibody, this was cultured for 48 hours. After culturing, morphological changes in the cells were observed under a
microscope. Viable cell count was determined by adding WST-8 (viable cell count reagent SF; Nacalai Tesque), cul-
turing at 37°C for two hours, and measuring OD 450 to measure the relative viable cell count.
[9] Induction of cell death by cross-linking
10
[0083] Jurkat cells were plated on a 24-well plate at 8 x 1 0 5 cells/well, and 1 0 |ig/ml of anti-mouse IgG (Fc) antibody
(Cappel) was further added in the presence (5 ng/ml) or absence of 2D7 antibody. 48 hours later, the cells were col-
lected, and after washing with PBS, methanol was added to a concentration of 70%, and this was left to stand at -20°C
for 15 minutes. After washing the cells with FACS buffer several times, Hoechst 33258 was added at a concentration
15 of 10 ng/ml, and this was incubated at room temperature for 30 minutes. The cells were washed again with FACS
Buffer, and then placed on a slide glass as a droplet to observe the state of the nuclei under a fluorescence microscope.
[1 0] Cloning of the 2D7 variable region
20 [0084] Total RNA was purified from 2D7 hybridoma (provided from University of Tokushima) using Trizol according
to standard methods. Using 3 |ig of this RNA as a template, cDNAs were synthesized using a SMART RACE cDNA
Amplification kit (CLONTECH), according to the attached instructions. Using this cDNA as a template, the variable
regions of the heavy chain and light chain were amplified by PCR using the following primers:
25 Heavy chain: 5'-CAGGGGCCAGTGGATAGACTGATG (SEQ ID NO: 9)
Light chain: 5'-GCTCACTGGATGGTGGGAAGATG (SEQ ID NO: 10)
[0085] The amplified cDNAs encoding each of variable regions were subcloned into pCR-TOPO vector (Invitrogen),
and the nucleotide sequences (SEQ ID NOs: 1 and 3) were determined.
30
[11] Production of 2D7 diabody expression vector
[0086] Plasmids, to which each of the variable region cDNAs were subcloned, were used as templates, and the
variable regions of the heavy chain and light chain (VH and VL) were respective amplified using the primers below:
35
Heavy chain
2D7DB-H1 : 5'-CCTGAATTCCACCATGCGATGGAGCTGGATCTTTC (SEQ ID NO: 11)
2D7DB-H2: 5'-AATTTGGCTACCGCCTCCACCTGAGGAGACTGTGAGAGTGGTGCCCT (SEQ ID NO: 12)
40
Light chain
2D7DB-L1 : 5'-TCCTCAGGTGGAGGCGGTAGCCAAATTGTTCTCACCCAGTCGCCAGC (SEQ ID NO: 13)
2D7DB-L2: 5'-ATTGCGGCCGCTTATCACTTATCGTCGTCATCCTTGTAGTCTTTTATCTCCAACTTTG TC-
45 CCCGAGCC (SEQ ID NO: 14)
[0087] Each of the VH and VL cDNAs amplified by these primers were combined into one tube, and further subjected
to PCR. Using the PCR products as templates, PCR was performed again, this time using 2D7DB-H1 and 2D7DB-L2
as primers, to synthesize cDNAwith VH and VL linked through a5-mer linker (SEQ ID NO: 5). This cDNAwas digested
so with EcoRI-Notl and inserted into the EcoRI-Notl gap of the animal cell expression vector, pCXND3. The nucleotide
sequence was confirmed, completing the construction of the 2D7 diabody expression vector, pCXND3-2D7DB.
[12] Transient expression in COS7 cells
55 [0088] 2 ng of pCXND3-2D7DB, or of an empty vector used as acontrol, was mixed with 6 |xL of transfection reagent
(LT-1, MIRUS) according to the attached instructions, and this was added to COS7 cells (plated the day before into a
6-well plate at 1x 10 5 cells/well) whose medium had been exchanged to a serum-free medium (OPTI-MEM, GIBCO
BRL). Five hours later, 200 |^L of serum was added, and this was cultured for two to three days. The medium was
13
EP 1 561 759 A1
collected, and dead cells were removed by centrifugation. The culture supernatant was then used for an experiment
to detect cytotoxic activity.
[0089] Expression of 2D7DB in the culture supernatant was confirmed by Western blotting. More specifically, equal
amounts of 2x SDS-PAGE Sample buffer and culture supernatant were added. In addition, after lysing the cells by
s adding a lysis buffer (0.5% Nonidet P-40, 1 0 mM Tris, pH 7.6, 1 50 mM NaCI, 5 mM EDTA), insolubilized proteins were
removed by centrifugation to prepare a cell lysate, and an equal amount of 2x SDS-PAGE Sample buffer was added
to this. After performing SDS-PAGE on each sample, the gels were transferred to PVDF membranes, and expression
of the 2D7 single chain was detected using anti-FLAG antibody.
10 [13] Establishment of expression cell lines producing 2D7 diabody
[0090] 20 \ig of pCXND3-2D7DB, linearized by cleaving with Pvul, was introduced to CHO cells (DXB11 strain) by
electroporation, as described below.
[0091] After washing the CHO cells twice with ice-cold PBS, they were suspended in PBS at 1x1 0 7 cells/ml. 20 |xg
15 of the above-mentioned plasmid was mixed into these cells, and this was electropulsed (1.5 KV, 25 nFD). The cells
were diluted in to appropriate fractions, plated on to a 10 cm dish, and cultured in the presence of G41 8 (GIBCO BRL)
at a final concentration of 50 ng/ml. Approximately 30 clones were selected from the grown colonies, and the diabody
expression levels in the culture supernatants were investigated by Western blotting. The clone with the highest ex-
pression level was expanded in a nucleic acid-free MEMoc medium containing 5 nM MTX, and this was stocked as an
20 overproducing cell line.
[14] Large-scale purification of 2D7 diabodies
[0092] A subconfluent 2D7DB-producing CHO cell line in a T-125 flask was detached using Trypsin-EDTA, and then
25 this was transferred to a roller bottle (250 ml of MEMoc without nucleotide + 5% FCS). Four days later, the culture
solution was removed, and the cells were washed twice with PBS. The medium was then exchanged to 250 ml of
CHO-S-SFMII medium (GIBCO BRL) to produce a serum-free medium, cells were cultured for three days, and then
the cell culture supernatant was collected. After removing the dead cells by centrifugation, this was filtered and used
for purification.
30 [0093] Purification of single chain Fv was performed as follows: First, the collected culture supernatant was applied
and adsorbed onto an anti-Flag M2 column. After washing with buffer A (50 mM Tris-HCI pH7.4, 150 mM NaCI, 0.01%
Tween 20), single chain Fv was eluted with buffer B (1 00 mM Glycine pH3.5, 0.01% Tween 20). The collected sample
was immediately neutralized with Tris-HCI pH8.0 so that the final concentration was 25 mM. This was then used for
gel filtration purification by a Superdex 200HR (26/60) column. The dimer fraction of single chain Fv was collected in
35 PBS containing 0.01% Tween 20. A portion of the collected sample was subjected to SDS electrophoresis and silver
staining to confirm that the protein of interest has been purified, and then this was concentrated to produce a purified
authentic sample of 2D7 diabody.
[15] Cell death induction experiment using 2D7 diabody
40
[0094] Various hemocyte cell lines were plated into 24-well plates at 2-5 x 10 5 cells/well. Purified 2D7DB, or the
culture supernatant of COS7 transiently expressing 2D7DB, was added and cell death was induced. When used, the
culture supernatant of COS7 transiently expressing 2D7DB was added so its concentration was 50%. The amount of
medium in each well was 0.8 to 1 ml/well. When stimulating Jurkat cells, Con A (WAKO) was added at the time of
45 2D7DB addition to a final concentration of 2 ng/ml.
[0095] Adherent cells (HeLa) were plated into a 6-well plate at 2x 10 5 cells/well, and the cells were attached by
culturing overnight. Subsequently, purified 2D7DB was added to the culture solution.
[0096] Several hours to several days after 2D7DB addition, the suspended cells were collected as they were, and
adherent cells were collected after detaching the cells with 1 mM EDTA/PBS. The cells were then washed with ice-
so cold PBS, and labeled with Annexin V, which is an apoptosis marker, and with PI, which is a dead-cell marker, according
to the attached instructions (TACS Annexin V-FITC Apoptosis Detection Kit, TREVIGEN Instructions). The proportion
of stained cells was then measured using flow cytometry (EPICS ELITE, COULTER).
[16] Cell death induction by Actinomycin D
55
[0097] Various hemocyte cell lines were plated into 24-well plates at 2-5 x 14 5 cells/well. To inhibit the initial stage
of apoptosis, a caspase inhibitor (Z-VAD-FMK, Promega) was added at a final concentration of 50 |^M, and after incu-
bating for 2.5 hours, cell death was induced. For cell death induction by Actinomycin D, Actinomycin D (Sigma) was
14
EP 1 561 759 A1
added at 1 ng/ml (Jurkat) or 5 ng/ml (ARH77), and for cell death induction by 2D7DB, 2 ng/ml of purified 2D7DB was
added. Cells were collected 1 6 hours after cell death induction, and stained using Annexin V and PI.
[17] Cell growth assay using 2D7 diabody
5
[0098] Each type of cells was plated into a 96-well plate at a cell concentration of 1-2 x10 4 cells/well. 2D7DB was
added at an appropriate concentration, and the cell count was determined after three days of culturing. Viable cell
count was determined using WST-8. More specifically, this reagent was added to the cells at 1 0 j^l/well, and the cells
were then cultured at 37°C for 1 .5 hours. The relative viable cell count was determined by measuring the OD 450 using
10 a spectrophotometer. The growth suppression rate was calculated from (1 - (OD 450 of 2D7DB treated cells / OD 450 of
2D7DB untreated cells)) x 100.
[18] Detection of DNA fragmentation
15 [0099] ARH77 and Jurkat cells were plated into a 6-well plate so that the cell concentration was 2 x 1 0 6 cells/well,
and cell death was induced by adding purified 2D7DB at a final concentration of 2 ng/ml, or Actinomycin D at a final
concentration of 1 ng/ml (ARH77) or 5 ng/ml (Jurkat) to each well. The control was a well to which nothing was added.
After culturing for 24 hours, the cells were collected, washed once with PBS, and then lysed in a lysis buffer (10 mM
Tris pH7.5, 10 mM EDTA, 0.5% Triton X-100). This was followed by centrifugation to remove the insoluble proteins,
20 and then the material was treated with RNase A and Proteinase K. A portion of this was then subjected to agarose gel
electrophoresis to detect chromatin DNA fragmentation.
[1 9] Inhibition of cell death induction by cytochalasin D
25 [0100] ARH77 cells were plated into a 24-well plate to achieve a cell concentration of 5 x 1 0 5 cells/well, and cytoch-
alasin D (Sigma) was added to a final concentration of 20 ng/ml- The control was a well to which ethanol alone was
added. After culturing for one hour, purified 2D7DB was added at various concentrations (0, 200, 500, 1000 ng/ml),
and culturing was continued for another four hours. Cells were then collected, and the proportion of dead cells was
detected by staining with PI.
30
[20] Immunostaining of 2D7DB-treated cells using anti-actin antibody
[0101] 2D7DB was added at a concentration of 1 ng/ml to cytochalasin D-treated/-untreated ARH77 cells, and after
culturing at 37°C for 15 minutes, the cells were adhered to a slide glass with Cytospin. After immobilizing the cells by
35 immersion in methanol for 15 minutes at -20°C, blocking was performed using a blocking buffer (3% BSA/PBS) at 4°C
for one hour. This was then reacted with CY3-labeled anti-actin antibody (Sigma) diluted 1 00-fold in 1 % BSA/PBS for
one hour at room temperature, and then the cell nuclei were stained with Hoechst 33258. After washing several times
with PBS, the cells were observed under aconfocal laser scanning microscope (Olympus).
40 [Example 1 ] Expression analysis of 2D7 antigen in each type of cell line
[0102] To determine the cell line that should become the source to produce a cDNA expression library and the cell
line that should become the host, 2D7 antigen expression in each type of animal cell was analyzed using FACS (Fig.
2A and Fig. 2B). As a result, among human-derived hemocyte cells, extremely strong expression of the 2D7 antigen
45 was observed in lymphocytic tumor cell lines, RPMI8226, U266, and in Jurkat, but expression was found to be weak
in K562. In Ba/F3, FDC-P1 , and HCI-1 6, which are hemocytes derived from mice, expression was very weak, perhaps
due to differences between the species. Of the adherent cells, expression was observed in COS7, 293T, and HeLa.
Expression was hardly observed in mouse NIH3T3 cells.
[0103] From the expression patterns mentioned above, RPMI8226 cells were judged to be appropriate as a source
so of a cDNA library to be used for expression cloning, and NIH3T3 cells were determined to be appropriate as host cells
to be used for screening, to which the expression library is transferred.
[Example 2] Cloning of 2D7 antigen
55 [1] Cloning from a protein
[0104] Cell lysates were prepared from RPMI8226 cells and U266 cells, which express the 2D7 antigen, and NIH3T3
cells, which do not express the 2D7 antigen, and immunoprecipitation was performed using the 2D7 antibody. As a
15
EP 1 561 759 A1
result, a molecule (approximately 12 kD) that precipitates specifically in RPMI8226 and U266 cells was observed (Fig.
3). This molecule was not detected by Western blotting using the 2D7 antibody, but since it is at least reproducibly
precipitated by the 2D7 antibody, it was strongly predicted to be the2D7 antigen itself, or a molecule that co-precipitates
with the 2D7 antigen.
s [0105] Coomassie staining was performed on this band; it was then cut out and the peptides were sequenced. As a
result, this 12 kD molecule was identified as fS2 microglobulin (p2M). Since p2M is one of the class I MHC protein
complexes that associate with HLA class I through non-covalent bonds, the 2D7 antibody is considered to have co-
precipitated it as an HLA complex. HLA class I comprises the oil and oc2 domains required for antigen presentation,
and the oc3 domain which binds to f!2M. Since the 2D7 antibody can co-precipitate the |32M molecule, it is anticipated
10 that the 2D7 antibody will recognize the oc1-oc2 domains of HLA class I as an epitope.
[2] Expression cloning of genes
[0106] cDNAs were synthesized using random hexamers from mRNAs purified from the 2D7 antigen-expressing
15 cells, RPMI8226. These were inserted into a retrovirus vector, pMX2, and a retrovirus expression library was construct-
ed. The library titer was investigated, and found to include a total of 6 x10 6 clones. Furthermore, the average cDNA
length was found to be approximately 1.5 kb, arrived at by randomly selecting 24 clones from this library and investi-
gating their insert size using colony PCR. Thus, the produced expression library was judged to be sufficient for use in
expression cloning.
20 [0107] Fig. 4A and Fig. 4B show a flow diagram of the screening described below. In the first screening, 4000 inde-
pendent clones were used in one pool, and 24 pools (corresponding to 96000 clones) were produced. The plasmids
were packaged into retroviruses by transfecting each plasmid into BOSC23 cells. The resulting viruses derived from
each pool were infected into NIH3T3 cells. Three days after infection, the cells were detached, and after staining with
2D7 antibody, expression analysis was performed using FACS. As a result, compared to NIH3T3 cells infected with
25 viruses derived from an empty vector (control), 2D7-positive cells were found in 3 of the 24 pools (pools 4, 1 3, and 21 ).
[0108] Next, pools 4 and 13, which showed positive results in the first screening, were divided into four pools each
comprising 1000 independent clones, and a second screening was performed. As a result, a single clearly positive
pool was found from each pool (Fig. 5A, pool 4-4, and pool 13-1). Pool 13-1 was further divided into 21 pools, each
comprising 160 independent clones, to perform a third screening. Two positive pools (Fig. 5B, 13-1-11 and 13-1-21)
30 were identified. Subsequently, pool 13-1-11 was divided into eight pools, each comprising 20 clones, to perform a fourth
screening, and a positive pool (Fig. 5C, 13-1-11-5) was obtained.
[0109] This pool was spread onto an LB plate, 64 colonies were picked one by one, and each of these were inoculated
to one well of a 96-well plate. The eight clones in the vertical rows were taken as one pool to produce eight pools (1
to 8), and the eight clones in the horizontal rows were taken as one pool to produce eight pools (A to H), and a fifth
35 screening was performed. As a result, pools 3, 4, 6, and 8, and pools E, F, and G were positive, thus narrowing down
the positive candidate clones to twelve clones (Fig. 6A). FACS was performed on these twelve clones, and ultimately
four positive clones (3F, 4G, 6E, and 8G) were identified as a single clone recognized by the 2D7 antibody (Fig. 6B).
[01 10] As a result of reading the sequence of the insert portion of these clones, all four clones were found to be the
full-length cDNA sequence of Human MHC class I HLA-A-6802.
40 [0111] HLA-A is classified into several dozen types of haplotypes. As a result of this cloning, the A*6802 haplotype
of HLA class I was identified as a 2D7 antigen, but since the 2D7 antibody recognizes a wide variety of cells, the
haplotype of HLA class I in the RPMI8226 cells that were used as the gene source just happened to be A*6802, and
the 2D7 antibody was considered to be an antibody that recognizes any haplotype of HLA class I molecules.
45 [Example 3] Examination of growth inhibitory effect
[01 12] Several types of leukemia cell lines (K562, Jurkat, and RPMI8226) were used to investigate whether the 2D7
antibody has a cytocidal effect. The expression level of the 2D7 antigen in the three cell lines is: K562, weakly positive;
Jurkat and RPMI8226, strongly positive.
so [0113] K562 and Jurkat cells were plated in the presence or absence of PHA and PMA, and 10 ng/ml of the 2D7
antibody was added thereto. On observing the cells 24 hours later, weakly 2D7-positive K562 cells did not show obvious
differences in their morphology due to the presence or absence of the 2D7 antibody, however, addition of 2D7 antibody
resulted in significant cell aggregation in Jurkat cells strongly expressing 2D7 (Fig. 7B and Fig. 7C). However, growth
inhibition due to addition of the 2D7 antibody was not observed (Fig. 7A). Growth inhibition due to 2D7 in Jurkat cells
55 activated by PHA and PMA stimulation was also not observed.
[0114] Unexpectedly, addition of 2D7 antibody did not have an obvious effect on the morphology and growth of the
strongly 2D7-positive RPMI8226 cells (Fig. 7D).
[0115] Next, it was examined whether cytocidal effects can be observed by adding anti-mouse IgG(Fc) antibody to
16
EP 1 561 759 A1
2D7 antibody, to cross-link the antibodies. Anti-mouse IgG was added to Jurkat cells, in the presence or absence of
2D7 antibody. The cells were cultured, and 48 hours later the cell nuclei were stained with Hoechst33258. Cells were
observed for fragmentation of cell nuclei, which is characteristic of dead cells (Fig. 8). As a result, nuclear fragmentation
was observed in Jurkat cells by further cross-linking 2D7 with an antibody, indicating that cell death was induced.
5
[Example 4] Cloning of cDNA encoding the 2D7 antibody variable region, and the predicted diabody structure
[01 16] Primers for the constant regions of the heavy chain and light chain of mouse lgG2b were produced, and DNA
encoding the 2D7 variable region was cloned by 5'RACE method. The nucleotide sequences of the obtained PCR
10 products are shown in SEQ ID NO: 1 and 3.
[0117] A single chain was then constructed based on these sequences. As shown in Fig. 9 and Fig. 10A, the 2D7
single chain is composed of the leader sequence of the heavy chain, the variable region of the heavy chain, and then
across from a 5mer linker (GGGGS), the variable region of a light chain, followed by a cDNA (SEQ ID NO: 5) encoding
a Flag-tag. Dimerization of this single chain may cause the 2D7 diabody to form the structure shown in Fig. 10B.
15
[Example 5] Analysis of the cytotoxic activity of the 2D7 diabody
(i) Cytotoxic activity of the 2D7 diabody transiently expressed in COS7
20 [01 18] A 2D7 diabody expression vector was transfected into COS7 cells, and the culture supernatant was collected
three days later. The culture supernatant and cell lysate were subjected to SDS-PAGE, and after performing Western
blotting with an anti-Flag-tag antibody, a2D7 single chain was found to be secreted in the culture supernatant (Fig. 1 0C).
[01 19] This culture supernatant was added to Jurkat cells at a ratio of 50%. The percentage of dead cells was meas-
ured by staining the cells with PI and Annexin V a few days later. No significant change in the apoptosis marker was
25 observed in Jurkat cells to which just the anti-BST-1 antibody and 2D7 antibody (5 ng/ml each) were added. Further-
more, no particular change could be observed when using the culture supernatant of COS7 transfected with the vector
alone. On the other hand, cell death was clearly induced in Jurkat cells to which the culture supernatant of COS7
expressing 2D7DB was added (Fig. 11 A and Fig. 11 B).
[0120] Next, to investigate the HLA class I A-specific action of this 2D7DB, a similar experiment was performed using
30 K562 cells, which are known to not express HLA class I A. As a result, 2D7DB had absolutely no influence on K562
cells, although it showed cell death inducing activity against Jurkat cells (Fig. 12A and Fig. 12B). This strongly supports
the idea that the cell death inducing activity of 2D7DB is an action targeting HLA class I A, which is its epitope. Fur-
thermore, according to each data, the sensitivity of Jurkat cells towards 2D7DB was found to be slightly higher in cells
stimulated with con A.
35 [0121] Next, the action of 2D7DB on other myeloma cell lines was analyzed. RPMI8226, IL-KM3, U266, and ARH77
were incubated with culture supernatant in which the vector alone was transfected (control), or with the 2D7DB-ex-
pressing COS7 culture supernatant. Two days later these cultures were double stained with Annexin V and PI, and
analyzed using a flow cytometer. As a result, incubation with 2D7DB was found to significantly induce cell death in all
of the cells (Fig. 13A to Fig. 13D).
40
(ii) Cytotoxic activity of purified 2D7DB
[0122] The growth inhibitory effect of purified 2D7DB on each type of cell line (RPMI8226, ARH77, U266, and Jurkat)
was analyzed. 2D7DB was added at 0, 0.5, 1 .0, and 2.0 ng/ml, and the number of cells was counted three days later.
45 As a result, 2D7DB was found to inhibit cell growth of these cells in a concentration-dependent manner (Fig. 14).
[0123] Purified 2D7DB was then added, and 48 hours later, cells were stained with cell death markers, PI and Annexin
V, and then analyzed. As in the results obtained when using 2D7DB transiently expressed in COS7, cell death was
induced in Jurkat and ARH77 in a concentration-dependent manner, and K562 was not affected at all (Fig. 15A to Fig.
15C). Furthermore, 48 hours after the addition of 2D7DB to U266 and IL-KM3, significant cell death inducing activity
so was confirmed (Fig. 16A and Fig. 16B).
[0124] On the other hand, although the 2D7 antibody stained the adherent HeLa cells very well, 2D7DB had abso-
lutely no influence under the same conditions (Fig. 15 D). This suggested that 2D7DB may act specifically on suspended
cells, such as hemocyte cells.
[0125] Next, the time taken for 2D7DB to induce cell death was analyzed. 2 jig/ml of 2D7DB was added to ARH77
55 and Jurkat cells, cells were collected 12, 24, and 38 hours later, and stained with a cell death marker. The results
showed that cell death was already induced in all cells twelve hours later (Fig. 1 7A and Fig. 1 7B). Therefore, cell death
induction was investigated at earlier times (three and six hours). Surprisingly, it was shown that 2D7DB induces cell
death at least within three hours after its addition (Fig. 1 8A and Fig. 1 8B). These results strongly support the idea that
17
EP 1 561 759 A1
2D7DB has a very strong cell death-inducing activity. Since 2D7DB strongly induces cell death, sufficient drug efficacy
can be expected even with a short half life in the blood. Furthermore, safety becomes a concern if the whole antibody
has strong cell death-inducing activity, considering the length of the half life in the blood; however, producing adiabody
can overcome such problems.
s [0126] Next, analyses were performed to determine whether cell death due to 2D7DB is induced through caspase
activation, that is, whether it is apoptosis. As shown in Fig. 19 and Fig. 20, significant apoptosis was induced when
ARH77 and Jurkat cells were treated with the apoptosis-inducing agent Actinomycin D, and then stained 1 6 hours later
with Annexin V and PI. After pre-treating cells under these conditions with caspase inhibitor Z-VAD-FMK for 2.5 hours,
apoptosis due to Actinomycin D was suppressed. However, cell death induced by 2D7DB was not inhibited at all by
10 pretreatment with Z-VAD-FMK. These results show that 2D7DB induces cell death by a mechanism different from the
ordinary caspase-mediated apoptosis mechanism.
[0127] To confirm this, fragmentation of chromatin DNA, known to be the most characteristic biochemical change
accompanying apoptosis, was also analyzed.
[0128] ARH77 and Jurkat cells were treated with 2D7DB (2 |xg/ml) or Actinomycin D, and DNAs were collected from
15 the cells 24 hours later and subjected to electrophoresis (Fig. 21). As a result, DNA fragmentation characteristic of
apoptosis had been induced in all cells treated with Actinomycin D, which is an apoptosis-inducing agent. On the other
hand, DNA fragmentation was not observed at all in 2D7DB-treated cells, even though the concentration of added
2D7DB was absolutely sufficient to induce cell death. These results also strongly support the idea that cell death due
to 2D7DB is an unknown type of cell death, unaccompanied by the characteristics of apoptosis.
20 [0129] From the above-mentioned results, cell death due to 2D7DB was found to be caused by a pathway different
from previously known cell death induction mechanisms. Therefore, further analysis was performed to elucidate the
mechanism of cell death induction by 2D7DB. From the experiments described above, when 2D7DB was reacted with
the cells, the cell membranes were often observed to be destroyed under the microscope. Therefore, 2D7DB was
presumed to have some sort of influence on the actin skeleton. In order to examine such a possibility, an actin polym-
25 erization inhibitor (cytochalasin D) was made to act on the cells, and the influence of 2D7DB on cell death induction
activity was analyzed.
[0130] Cytochalasin D (20 ng/ml) or ethanol alone (control) was added to ARH77 cells, and 1 hour later, 2D7DB was
added at various concentrations. After a 4-hour incubation from the 2D7DB addition, cells were collected, PI staining
was performed and the percentage of dead cells was measured (Fig. 22). As a result, pretreatment of cells with cyto-
30 chalasin D was found to cause loss of sensitivity towards 2D7DB. These results suggested that 2D7DB causes some
kind of effect on the cytoskeletal system, such as actin, to induce cell death by binding to HLA-class IA, which is the
target molecule.
[0131] Therefore, cells treated with 2D7DB were stained by the actin antibody, and the dynamic change of the cy-
toskeletal system due to 2D7DB addition was analyzed visually. ARH77 cells were treated with 2D7DB, and 1 5 minutes
35 later, the cells were immobilized with methanol, and the state of actin (red) in the cells was investigated by immunos-
taining (Fig. 23). As a result, compared to the image from those not treated with 2D7DB, significant destruction of the
actin skeleton in the cell due to 2D7DB was observed.
[0132] The above-mentioned results strongly suggested that cell death due to 2D7DB may be caused by destruction
of the actin skeleton in cells by 2D7DB bound to HLA class IA. This is a completely new type of cell death induction
40 mechanism that has not been reported to date.
[Example 6] Drug efficacy test for 2D7 diabody using human myeloma animal model
(1) Production of mouse model for human myeloma
45
[0133] A mouse model for human myeloma was produced as follows. ARH 77 cells were prepared to reach 2.5x 1 0 7
cells/ml in RPMI1640 medium (GIBCO BRL) supplemented with 10% fetal calf serum (GIBCO BRL), and then 200 |xL
of the above-mentioned ARH77 cell suspension (5x 10 6 cells/mouse) was injected to SCID mice (male, 6 weeks old,
Clea Japan) pretreated the day before with intraperitoneal administration of 0.2 mg of anti-asialo GM1 antibody (Wako
so Pure Chemicals) from the tail vein.
(2) Preparation of the antibody to be administered
[0134] On the day of administration, a 2D7 diabody was prepared at 0.8 mg/ml using filter-sterilized PBS(-), and this
55 was used as the administration sample.
18
EP 1 561 759 A1
(3) Antibody administration
[0135] To the mouse model of human myeloma produced in (1), the administration sample prepared in (2) was
administered through the tail vein at 1 0 ml/kg twice a day for 3 days from the first day after engraftment of ARH77 cells.
s As a negative control (vehicle), filter-sterilized PBS(-) was administered similarly at 1 0 ml/kg through the tail vein twice
a day for 3 days. The antibody-administered group had 7 animals per group, and the vehicle-administered group had
8 animals per group.
(4) Human IgG assay of mouse serum
10
[0136] The quantity of human IgG produced by human myeloma cells in the mouse serum was determined by ELISA
described below. 100 [A. of goat anti-human IgG antibody (BIOSOURCE) diluted to 1 |xg/ml with 0.1% bicarbonate
buffer (pH9.6) was placed into a 96-well plate (Nunc), this was incubated at 4°C overnight, and the antibody was
immobilized. After blocking, mouse serum diluted in a stepwise manner, or as the authentic sample, 1 00 \jlL of human
15 IgG (Cappel) was added, and this was incubated at room temperature for 1 hour. After washing, 1 00 \jlL of a 5000-fold
diluted alkaline phosphatase-labeled anti-human IgG antibody (BIOSOURCE) was added, and this was incubated at
room temperature for 1 hour. After washing, substrate solution was added, and after incubation, absorbance at 405
nm was measured using MICROPLATE READER Model 3550 (BioRad), and the concentration of human IgG in mouse
serum was calculated from the calibration curve obtained from the absorbance of the authentic human IgG sample.
20
(5) Evaluation of anti-tumor effect
[0137] The anti-tumor effect of the 2D7 diabody on a human myeloma mouse model was evaluated using the change
in the amount of human IgG (M protein) produced by the myeloma cells in mouse serum, and by the survival time.
25 Regarding the change in human IgG level in mouse serum, serum was collected on the 24th day after transplanting
the ARH77 cells, and the human IgG level was measured by the ELISA described above in (4). As a result, the level
of human IgG (M protein) in the serum had increased in the vehicle-administered group to approximately 74 ng/ml. In
contrast, the level in the 2D7 diabody-administered group was significantly lower than in the control group (P<0.005,
unpaired t-test), and 2D7 diabody was shown to very strongly suppress the growth of ARH77 cells (Fig. 24). With
30 regards to survival time, as shown in Fig. 25, the 2D7 diabody-administered group showed a significant increase in
survival time compared to the vehicle-administered group.
[0138] Accordingly, the 2D7 diabody was shown to have an antitumor effect on the mouse model of human myeloma.
The antitumor effect of the 2D7 diabodies of this invention may be based on the cell death-inducing action of this
antibody.
35
[Example 7] Analysis of the action of 2D7DB on PBMC
[0139] The action of 2D7DB on human peripheral blood mononuclear cells (PBMCs) was analyzed. PBMCs were
purified from the peripheral blood of a healthy adult volunteer by density gradient centrifugation. The PBMCs were
40 plated at 5x 1 0 5 cells/1 ml/well onto a 24-well plate, in the presence or absence of a mitogen. Phytohemagglutinin M
(PHA-M, Roche Diagnostics, final concentration: 10 |^g/ml), concanavalin A (ConA, Wako, final concentration: 10 |^g/
ml), and SAC (Pansorbin Cells, Calbiochem, final concentration: 0.01%) were used as mitogens. Cells were cultured
in a 5% C0 2 incubator at 37°C for three days. 24 or 3 hours before culture was complete, 2D7DB was added to yield
a final concentration of 2 ng/ml. After culture was complete, the cells were double stained with Annexin V and PI
45 (Annexin V-FITC Apoptosis Detection Kit I, Pharmingen), and then analyzed using a flow cytometer (EPICS XL, Coul-
ter). As a positive control, ARH77 at 2.5x1 0 5 cells/1 ml/well was cultured for 24 hours in the absence of a mitogen,
and was reacted with 2D7DB, as for PBMC.
[0140] In the case of PBMC, the percentages of dead cells that were both Annexin V and Pl-positive were 29%,
23%, and 25% in the absence of mitogens (in order: no addition, 3-hour addition, and 24-hour addition of 2D7DB;
so continued below); 20%, 45%, and 42% in the presence of PHA-M; 22%, 30%, and 34% in the presence of ConA; and
31%, 38%, and 40% in the presence of SAC (Figs. 26A to 26D). In the case of ARH77, the percentages were 16%,
56%, and 58% (Fig. 26E). These results showed that2D7DB has hardly any effect on unstimulated PBMC, but induces
cell death in a short time with mitogen-activated PBMC.
55 Industrial Applicability
[0141] This invention provides minibodies with high specific activities. By using these minibodies, adequate drug
efficacy can be expected even with a short half life. The minibodies of the present invention are further expected to be
19
EP 1 561 759 A1
able to separate drug efficacy from toxicity. In addition, since overall cost is reduced, including reducing clinical dose
and production cost, economical problems of concern in the development of antibody pharmaceuticals are also ex-
pected to improve.
20
EP 1 561 759 A1
SEQUENCE LISTING
<110> CHUGAI SEIYAKU KABUSHIKI KAISHA :
OZAKI Shuji
ABE Masahiro
<120> Cell Death-Inducing Agent
<130> C1-A0220P
<140> PCT/JP03/13063
<141> 2003-10-10
<150> JP 2002-299289
<151> 2002-10-11
<160> 14
<170> Patentln Ver. 2. 1
<210> 1
<211> 547
<212> DNA
<213> Mus musculus
<220> .
21
10
15
EP 1 561 759 A1
<221> CDS
<222> (103).. (546)
<400> 1
tacgactcac tatagggcaa gcagtggtat caacgcagag tacgcgggga atctatgatc 60
agtgtcctct ctacacagtc cctgacgaca ctgactccaa cc atg cga tgg age 114
Met Arg Trp .Ser'
1
tgg ate ttt etc ttc etc ctg tea ata act gca ggt gtc cat tgc cag 162
Trp lie Phe Leu Phe Leu Leu Ser He Thr Ala Gly Val His Cys Gin
5 10 15 20
gtc cag ttg cag cag tct gga cct gag ctg gtg aag cct ggg get tea 210
35 Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
25 30 35
20
40
45
50
gtg aag atg tct tgt aag get tct ggc tac ace ttc aca gac tac ttt 258
Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Phe
40 45 50
ata cac tgg gtg aaa cag agg cct gga cag gga ctt gaa tgg att gga 306
He His Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp lie Gly
55 60 65
22
EP 1 561 759 A1
tgg att ttt cct gga gat gat act act gat tac aat gag aag ttc agg 354
Trp lie Phe Pro Gly Asp Asp Thr Thr Asp Tyr Asn Glu Lys Phe Arg
70 75 80
ggc aag acc aca ctg act gca gac aaa. tec tec. age aca gec tac att 402
Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr He
85 90 95 100
ttg etc age age ctg acc tct gag gac tct gcg atg tat ttc tgt gta 450
Leu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Met Tyr Phe Cys Val
105 110 . 115.
agg agt gac gac ttt gac tac tgg ggc cag ggc acc act etc aca gtc 498
Arg Ser Asp Asp Phe Asp Tyr Trp Gly Gin Gly Thr Thr Leu Thr Val
120 125 130
tec tea gee aaa aca aca ccc cca tea gtc tat cca ctg gee cct get g 547
Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Ala
135 140 145
<210> 2
<211> 148
<212> PRT
<213> Mus musculus
23
EP 1 561 759 A1
<400> 2
Met Arg Trp Ser Trp lie Phe Leu Phe Leu Leu Ser lie Thr Ala Gly
1 5 10 15
Val His Cys Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys
20 25 30
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
Thr Asp Tyr Phe He His Trp Val Lys Gin Arg Pro Gly Gin Gly Leu
50 55 60
Glu Trp He Gly Trp He Phe Pro Gly Asp Asp Thr Thr Asp Tyr Asn
65 70 75 80
Glu Lys Phe Arg Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser
85 90 95
Thr Ala Tyr He Leu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Met
100 105 110
Tyr Phe Cys Val Arg Ser Asp Asp Phe Asp Tyr Trp Gly Gin Gly Thr .
115 120 125
Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro
24
EP 1 561 759 A1
130 135 140
Leu Ala Pro Ala
145
<210> 3
<211> 535 '
<212> DNA
<213> Mus musculus
<220>
<221> CDS
<222> (103). . (534)
<400> 3
ctaatacgac. tcactatagg gcaagcagtg gtatcaacgc agagtacgcg gggactwatg 60
agaatagcag taattagcta gggaccaaaa ttcaaagaca aa atg cat ttt caa 114
. Met His Phe Gin
1
gtg cag att ttc age ttc ctg eta ate agt gee tea gtc ate atg tec 162
Val Gin He Phe Ser Phe Leu Leu He Ser Ala Ser Val He Met Ser
5 10 15 20
25
EP 1 561 759 A1
aga gga caa att gtt etc acc cag teg cca gca ate atg tct gca tct
Arg Gly Gin He Val Leu Thr Gin Ser Pro Ala He Met Ser Ala Ser
25 30 35
cca ggg gag aag gtc acc ata acc tgc agt gee age tea agt gta agt
Pro Gly Glu Lys Val Thr lie Thr Cys Ser Ala Ser Ser Ser Val Ser
40 45 50
tac atg cac tgg ttc cag cag aag cca ggc act ttt ccc aaa etc tgg
Tyr Met His Trp Phe Gin Gin Lys Pro Gly Thr Phe Pro Lys Leu Trp
55 60 65
att tat age aca tec aac ctg get tct gga gtc cct act cgc ttc agt
He Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Thr Arg Phe Ser
70 75 80
ggc agt gga tct ggg acc tct tac tct etc aca ate age cga atg gag
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr He Ser Arg Met Glu
85 90 95 100
get gaa gat get gee act tat tac tgc cag caa agg acg agt tat cca
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Arg Thr Ser Tyr Pro
105 110 115
ccc acg ttc ggc teg ggg aca aag ttg gag ata aaa egg get gat get
Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu He Lys Arg Ala Asp Ala
26
EP 1 561 759 A1
- 120 125 130
gca cca act gta tec ate ttc cca. cca tec agt gag c
Ala Pro Thr Val Ser He Phe Pro Pro Ser Ser Glu
135 140
<210> 4
<211> 144 •
<212> PRT ■
<213> Mus musculus
<400> 4
Met His Phe Gin Val Gin He Phe Ser Phe Leu Leu lie Ser Ala Ser
1 5 10 15
Val lie Met Ser Arg Gly Gin He Val Leu Thr Gin Ser Pro Ala He
20 25 30
Met Ser Ala Ser Pro Gly Glu Lys Val Thr He Thr Cys Ser Ala Ser
35 40 . 45
Ser Ser Val Ser Tyr Met His Trp. Phe Gin Gin Lys Pro Gly Thr Phe
50 55 60
Pro Lys Leu Trp He Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro
27
EP 1 561 759 A1
65 ' 70 • 75 . 80
Thr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr lie
85 90 95
Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Arg
100 105 110
Thr Ser Tyr Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu He Lys
115 120 125
Arg Ala Asp Ala Ala Pro Thr Val Ser lie Phe Pro Pro Ser Ser Glu
130 135 140
<210> 5
<211> 789
<212> DNA
<213> Artificial Sequence
<220>
<221> CDS
<222> (14). . (775)
<223> Description of Artificial Sequence:an artificially
synthesized DNA sequence-
28
EP 1 561 759 A1
<400> 5
cctgaattcc acc'atg cga tgg age tgg ate ttt etc ttc etc ctg tea
Met Arg Trp Ser. Trp lie Phe Leu Phe Leu Leu Ser
1 5 10
ata act gca ggt gtc cat tgc cag gtc cag ttg cag cag tct gga cct
lie Thr Ala Gly Val His Cys Gin Val Gin Leu Gin. Gin Ser Gly Pro
15 20. 25
gag ctg gtg aag cct ggg get tea gtg aag atg tct tgt aag get tct
Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser
30 35 40
ggc tac ace ttc aca gac tac ttt ata cac tgg gtg aaa cag agg cct
Gly Tyr Thr Phe Thr Asp Tyr Phe He His Trp Val Lys Gin Arg Pro
45 50 55 60
gga cag gga ctt gaa tgg att gga tgg att ttt cct gga gat gat act
Gly Gin Gly Leu Glu Trp He Gly Trp He Phe Pro Gly Asp Asp Thr
65 70 75
act gat tac aat gag aag ttc agg ggc aag acc aca ctg act gca gac
Thr Asp Tyr Asn Glu Lys Phe Arg Gly Lys Thr Thr Leu Thr Ala Asp
80 85 90
aaa tec tec age. aca gee tac att ttg etc age age ctg acc tct gag
29
10
15
20
EP 1 561 759 A1
Lys Ser Ser Ser Thr Ala Tyr lie Leu Leu Ser Ser Leu Thr Ser Glu
'95 .100 105
gac tct gcg atg tat ttc tgt gta agg agt gac gac ttt gac tac tgg 385
Asp Ser Ala Met Tyr Phe Cys Val Arg Ser Asp Asp Phe Asp Tyr Trp
110 115 120
ggc cag ggc acc act etc aca gtc tec tea ggt gga ggc ggt age caa 433
Gly Gin Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gin
125 130 135 140
att gtt etc acc cag teg cca gca ate atg tct gca tct cca ggg gag 481
He Val Leu Thr Gin Ser Pro Ala He Met Ser Ala Ser Pro Gly Glu
145 150 155
aag gtc acc ata acc tgc agt gec age tea agt gta agt tac atg cac 529
35 Lys Val Thr He Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His
160 . 165 170
40
tgg ttc cag cag aag cca ggc act ttt ccc aaa etc tgg att tat age 577
Trp Phe Gin Gin Lys Pro Gly Thr Phe Pro Lys Leu Trp He Tyr Ser
45 175. 180 185
50
aca tec aac ctg get tct gga gtc cct act cgc ttc agt ggc agt gga 625
Thr Ser Asn Leu Ala Ser Gly Val Pro Thr Arg Phe Ser Gly Ser Gly
190 . 195 200
30
EP 1 561 759 A1
tct ggg ace tct tac .tct etc aca ate age cga atg gag get gaa gat
Ser Gly Thr Ser Tyr Ser Leu Thr lie Ser Arg Met Glu Ala Glu Asp
205 210 215 220
get gec act tat tac tgc cag caa agg acg agt tat cca ccc acg ttc
Ala Ala Thr Tyr Tyr Cys Gin Gin Arg Thr Ser Tyr Pro Pro Thr Phe
225 ' 230 235 "
ggc teg ggg aca aag ttg gag ata aaa gac tac aag gat gac gac gat
Gly Ser Gly Thr Lys Leu Glu. lie Lys Asp Tyr Lys Asp Asp Asp Asp
240 245 250
aag tga taagcggccg caat
Lys
<210>. 6
<211> 253
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:an artificially
synthesized peptide sequence
31
EP 1 561 759 A1
<400> 6
Met Arg Trp Ser Trp He Phe Leu Phe Leu Leu Ser He Thr Ala Gly
1 5 10 15
Val His Cys Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys
20 25 30
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
Thr Asp Tyr Phe lie His Trp Val Lys Gin Arg Pro Gly Gin Gly Leu
50 55 60
Glu Trp He Gly Trp lie Phe Pro Gly Asp Asp Thr Thr Asp Tyr Asn
65 70 75 . 80
Glu Lys Phe Arg Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser
85 90 95
Thr Ala Tyr He Leu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Met .
100 105 . lib
Tyr Phe Cys Val Arg Ser Asp Asp Phe Asp Tyr Trp Gly Gin Gly Thr
115 120 125 .
Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gin He Val Leu Thr
130 135 140
Gin Ser Pro Ala He Met Ser Ala Ser Pro Gly Glu Lys Val Thr He
145 150 155 160
Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Phe Gin. Gin
165 170 175
Lys Pro Gly Thr Phe Pro Lys Leu Trp He Tyr Ser Thr Ser Asn Leu .
180 185 190
Ala Ser Gly Val Pro Thr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser
32
EP 1 561 759 A1
.195 200 ... 205
Tyr Ser Leu Thr lie Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr
210 215 220
Tyr Cys Gin Gin Arg Thr Ser Tyr Pro Pro Thr Phe .Gly Ser Gly Thr
225 230 235 240
Lys Leu Glu lie Lys Asp Tyr Lys Asp Asp Asp Asp Lys
245 250
<210> 7
<211> 29
<212> DNA
<213> Artificial Sequence .
<220>
<223> Description of Artificial Sequence^an artificially
synthesized adapter sequence
<400> 7
aattcccagc acagtggtag ataagtaag
<210> 8
<211> 29
<212> DNA
<213> Artificial Sequence
33
EP 1 561 759 A1
10
15
<220>
<223> Description of Artificial Sequence :an artificially
synthesized adapter . sequence
<400> 8
tcgacttact tatctaccac tgtgctggg .29
20 <210> 9
<211> 24
<212> DNA
25
<213> Artificial Sequence
30
<220>
<223> Description of Artificial Sequence:an artificially
35 synthesized primer sequence
40
45
50
<400> 9
caggggccag tggatagact gatg. 24
<210> 10
<211> 23
<212> DNA
<213> Artificial Sequence
34
EP 1 561 759 A1
w
15
<220>
<223> Description of Artificial Sequence: an artificially-
synthesized primer sequence
<400> 10
gctcactgga tggtgggaag atg . . 23
20 <210> 11
<211> 35
<212> DNA .
25
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:an artificially
35 synthesized primer sequence
<400> 11
40 ■
cctgaattcc accatgcgat ggagctggat ctttc 35
45 . .
<210> 12
so <211> 47
<212> DNA
<213> Artificial Sequence
35
10
15
20
EP 1 561 759 A1
<220>
<223> Description of Artificial Sequence: an artificially
synthesized primer sequence
<400> 12
aatttggcta ccgcctccac ctgaggagac tgtgagagtg gtgccct 47
35
<210> 13
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:an artificially
synthesized primer sequence
<400> 13
40
tcctcaggtg gaggcggtag ccaaattgtt ctcacccagt cgccagc 47
45 • ...
<210> 14
50 <211> 68
<212> DNA
<213> Artificial Sequence
36
EP 1 561 759 A1
10
<220>
<223> Description of Artificial Sequence tan artificially
synthesized primer sequence
<400> 14
attgcggccg cttatcactt atcgtcgtca tccttgtagt cttttatctc caactttgtc 60
w cccgagcc
68
20
Claims
I. A minibody that recognizes a human leukocyte antigen (HLA).
25 2. The minibody of claim 1 , wherein the HLA is an HLA class I.
3. The minibody of claim 2, wherein the HLA class I is an HLA-A.
4. A minibody derived from a 2D7 antibody.
30
5. The minibody of any one of claims 1 to 4, wherein the minibody is a diabody.
6. A minibody of any one of (a) to (d):
35 (a) a minibody comprising the amino acid sequence of SEQ ID NO: 6;
(b) a minibody functionally equivalent to the minibody of (a), and comprising an amino acid sequence with a
substitution, insertion, deletion and/or addition of one or more amino acids in the amino acid sequence of SEQ
ID NO: 6;
(c) a minibody comprising the amino acid sequences of CDRs of SEQ ID NOs: 2 and 4; and
40 (d) a minibody functionally equivalent to the minibody of (c), and comprising an amino acid sequence with a
substitution, insertion, deletion and/or addition of one or more amino acids in the amino acid sequence of the
CDRs of SEQ ID NOs: 2 and 4.
7. A method for producing an HLA-recognizing antibody having increased activity by converting the HLA-recognizing
45 antibody to a low-molecular-weight antibody.
8. The method of claim 7, wherein the HLA is an HLA class I.
9. The method of claim 8, wherein the HLA class I is an HLA-A.
50
10. A method for producing a2D7 antibody having increased activity by converting the 2D7 antibody to alow-molecular-
weight antibody.
I I . The method of any one of claims 7 to 10, wherein the conversion step comprises conversion to a diabody.
55
12. The method of any one of claims 7 to 11, wherein the activity is a cell death-inducing activity or a cell growth-
suppressing activity.
37
EP 1 561 759 A1
13. A cell death-inducing agent, comprising as an active ingredient the minibody of any one of claims 1 to 6, the
minibody produced by the method of any one of claims 7 to 12, or a 2D7 antibody.
14. The cell death-inducing agent of claim 13 that induces cell death of a B cell or T cell.
5
15. The cell death-inducing agent of claim 14, wherein the B cell or T cell is an activated B cell or activated T cell.
16. A cell growth-suppressing agent comprising as an active ingredient the minibody of any one of claims 1 to 6, the
minibody produced by the method of any one of claims 7 to 12, or a 2D7 antibody.
10
17. An antitumor agent comprising as an active ingredient the minibody of any one of claims 1 to 6, the minibody
produced by the method of any one of claims 7 to 1 2, or a 2D7 antibody.
18. The antitumor agent of claim 1 7, wherein the tumor is a blood tumor.
15
19. A therapeutic agent for an autoimmune disease, wherein the therapeutic agent comprises as an active ingredient
the minibody of any one of claims 1 to 6, the minibody produced by the method of any one of claims 7 to 1 2, or a
2D7 antibody.
20 20. The cell death-inducing agent of any one of claims 13 to 15, wherein the antibody is a diabody.
21 . The cell growth-suppressing agent of claim 1 6, wherein the antibody is a diabody.
22. The antitumor agent of claim 1 7 or 1 8, wherein the antibody is a diabody.
25
23. The therapeutic agent for autoimmune disease of claim 1 9, wherein the antibody is a diabody.
38
EP 1 561 759 A1
FIG. 1
5 ' -AATTCCCAGCACAGTG GTAGATAA G TAA G (SEQ ID N0:7)
GGGTCGTGTCACC ATC T ATT C ATT CAGCT-5 ' (SEQ ID N0:8)
39
EP 1 561 759 A1
FIG. 2A
116 87 58 29 0 ' 85 63 42 21 0 '
40
EP 1 561 759 A1
FIG. 2B
o
O
134 100 67 33 0 "
o
C3
O
129 96 64 32 0 '
o
o
o
o
94 70 47 23 0 '
o
o
o
131 98. 65. 32 0 '
o
C3
O
O
108 81 54 27 0 '
41
EP 1 561 759 A1
FIG. 3
NIH3T3 RPMI8226 U266
42
EP 1 561 759 A1
FIG. 4 A
FIG. 4B
CM I
CO |
X
CO
LU
z:
o
i -
o
u_ o uj o
O «D 00
C3
5
LU
LU
UJ
OS
C3
CJ
CO
oo
C3
: —
oo
O
q:
LU
Ll.
co
43
EP 1 561 759 A1
44
EP 1 561 759 A1
FIG. 5B
1 i 1 1 mm i 1 1 TTtTl i i muti — i 1 1 unit
" 1 fitc 1000
1 1 iTTTO — i i i iiiiii — i 1 1 mil l
FITC 1000
o
13-1-3
o
i Tf mn i i i inn — 1 1 1 inn
fitc 1000
13-1-10
i 1 1 ii i iii i niiiiii i 1 1 nmi — i i nun 1
1 fitc 1000
FITC
ra — i i nun
1000
"i i n mil — r TTTTnn — f i iiimi — i i iiiiii
■\ fitc 1000
45
EP 1 561 759 A1
FIG. 5C
10 100 1000
FITC
1 — i i i 1 1 1 hi — i rm mi — iiii nm — j i i i mi 1
-1 1 10 100 1000
FITC
-1
10 100 1000
FITC
10 100 1000
FITC
A 13-1-11-3
'V i i i hi in — i i it i j iil " " f "i 1 1 1 iiii — i I I 1 1 ill
■1 1 10 100 1000
FITC
i i I nun 1 i 1 1 inn™ i i in iiii 1 mi mil
1 10 100 1000
FITC
13-1-11-5
.•■■.i-r; s j; > .
iii i ■ i m • i i 1 1 1 mi i i rim ii^ — i 1 1 iiii
1 10 100 1000 -1
FITC
13-1-11-6
i 1 1 nm — i i i n iiir i i 1 1 nm — iiii iiii
1 10 100 1000
FITC
o
13-1-1 1-7
T
i i i imn — r
1
t i nTfil' I i i nun i i i 1 1 in
10 100 1000
FITC
13-1-11-8
10 100 1000
FITC
46
EP 1 561 759 A1
FIG. 6A
COUNT
COUNT
COUNT
t — i — r
COUNT
COUNT
COUNT
"i — i — r
COUNT
i — i — r
COUNT
COUNT
COUNT
COUNT
E O
COUNT
-i — r
COUNT
COUNT
COUNT
COUNT
47
EP 1 561 759 A1
FIG. 6B
1 10 100 1000
FITC
1 10 100 1000
FITC
1 10 100 1000
FITC
1 10 100 1000
FITC
48
EP 1 561 759 A1
FIG. 7A
2.5
§ 1.5
1 -
0.5 -
□ PMA(-)2D7(-)
■ PMA(-)2D7(+)
■ PMA(+)2D7(-)
HPMA(+)2D7(+)
FIG. 7B
K562 Jurkat RPMI8226
FIG. 7C
- • -
• r ii J ■
$. i Jl i
u - -
%8»-^K%> : •' "■' "3;'- "rsUfi"
j
PHA/PMA(+)2D7(-)
PHA/PMA(+)2D7(+)
FIG. 7D 2E2Si
2D7(+)
49
EP 1 561 759 A1
FIG. 8
2D7 (-) ANT I -MOUSE IgG (-) 2D7(-) ANT I -MOUSE IgG (+)
2D7(+) ANT I -MOUSE IgG (-) 2D7(+) ANT I -MOUSE IgG <-)
50
EP 1 561 759 A1
FIG. 9
o c_D
o <C
•-t CD EQ
Eh
O
CJ Cu
<
(J
cd cd
Eh
O O
o-i e-h to
c_> o
cj
CD
E-h
O E->
m LD
^ _
cj o
o
E-"
CD
8
o cj
r- CD
sa
■ oi
cj
E-i.
CD >
Eh
o CD
vo o CD
• «=c
<-> ^
CD rij
Eh
CJ
<; Eh
cj
cd
cj
Eh
cd
Eh
cj
CJ
Eh
o
cj
Eh
Eh
CJ
Eh
CJ
Eh
Eh
Eh
CJ
S3 ►
CD
CD
Eh E
CJ
cd
< c
CD
CD
5 =
CD
CJ (
cd
S'
o
ro
CJ
CJ
4->
4-1
fT3
CO
C7<
EH
o
o
C3
< CJ
<-H < S
o
CD
o
<
ro
CJ
CJ
Eh
o
CJ
Eh
o
CJ
<
cd
<;
cj
CD
Eh
CJ
<
cd
3
CO
Eh
CM
CJ
CJ
<
o
CJ
CD
O
P~
CN]
CJ
cd
CD
CD
CD
6
Eh
o
Eh
CO
CD
CN]
CD
<
CD
Eh
o
CJ
<
C\l
Eh
%
CD
Eh
CJ
«<
Eh
o
CJ
<c
CN]
CD
Eh
3
<
CD
CD
CD
ro
Eh
CN]
CJ
CJ
Eh
Eh
EH
Eh
§
o
CM
CD
CM
13
CD
CD
Eh
s
CD
CD
CD
I — 1
CM
s
CD
Eh
Eh
CJ
<
CD
Eh
CD Eh
CD CJ
-=r j<
CJ
O
<
CJ
cd
CD CD
CD
CD <
CTv CJ O
m cj
CD
CD CD
CD
CD
Eh
%
CD Eh
CO CJ
ro cC
CD
Eh
Eh
• Eh
5*
CD
co CD
EH
a
CD
CD
CO
3
OS
CJ
O Eh
ko CD
ro Eh
. CD
Eh
CJ
Eh
Eh
O Eh >H
LO CD
ro Eh
< s
CD
CJ
CD <<
Eh
CJ
Eh CO
CJ
- fiC
ro CD Q
.a
CD W
Eh
CJ
Eh
CJ
CD CJ
ro <; Eh
ro CD
EH
CJ i-a
CJ
8
S3
CD
CM CJ
CO EH
CJ
CD
EH
Eh
Eh
og
i— i r£
Eh
CJ
O
1
CO
CO
CO
ro
CJ;
3
CD U
ro cn
-a" ro CO
3
o
CD
CD
r—
as
CJ
o
<5
CJ
o».
u
cd
CD
_
Eh
4->
CD
CJ
CD
<
rO
Eh
?H
O
Eh
«C
U
Eh
>-l
O
EH
CT>
CJ
■ ■
o>
o
<
EH
O
CO
CJ
CO
CP
CJ
I
ro
CD
CO
-
Eh
4->
CJ
nj
CD
o>
EH
4->
<
Cn
CD
(— 1
ro
cd
m «
r-
<
c
4->
CO
CD
CO
Eh
cji o
CJ
o
CD
CO '
CD
Cn Q
<
o
CD
CO
CD
cn Q
CD
Eh
4_)
<;
CO
CO
cn Q
CD
Cn
CJ
fX
ro
CJ
CO «
CD
o
<
CO
CO
CJ
4-> >H
Eh
o
CD
<
I — 1
CD
CO
lO
<
Cn Q
CO
[ —
<
t— <
CJ
Eh
CJ
i— J
Eh
Eh
et; i—i
CJ
CD
Eh
LO
<
o
O
CD
CD U
<
CD .
IT)
Eh
>-<
r~
Eh
Eh
Eh l-H 1
CJ
CD
Eh
CO
^
CJ
CJ
^
<
fc— *
CJ
CD
< Eh
CD
CD
o
CD
ro
CD
CD
ro
CD
Eh
r~
CD CD
CJ
CD
Eh
CO
CJ
<
Eh CO
CD
CJ
CD
CD
CD
Eh
CD CD
S3
CJ
6
CO
o
Eh
rsi
CM
Eh t-i
cc
CD
c—
CD
CD
CD
CJ
Eh
<! Eh
CJ
CO
CJ
s
CJ pu
Eh
6
Eh
Cu
o
CJ
CD
CJ Oj
<— i
CD
t— i
CD
CJ
OS
r-
%
Eh
Eh >->
Eh
H
Eh
CD
<C CO
CJ
CD
CJ
CJ
CJ
< H
51
EP 1 561 759 A1
FIG. 10A
LEADER SEQUENCE
FIG. 1 0B
52
EP 1 561 759 A1
FIG.11A
•a
£2
o
CD
o
CD
CM
o ■
-".V.-l *
CM
1 1 I I I [HIM I I I jlllll IT
O -O o
PI Log
E
o
CO
V3
CO
esi<<t .- • • -'. ' T - ' .
o •■■ . .-
3^" ;
o
o" 5
rt £ -'*--• •
o 00 •
o
O
_ o
o o
*~ PI Log
o
"■"
-. v *■■
o->- =
■VY:V •
tnrrrt 1 ( |iimi i
i — pin 1 1
|iuii i i r
_ o
o
i
P
7" PI Log
c
o
o
-TO
O
'• • ' ' ■ . . . ■' •"■ •<
=■ o
o
I
O
o o
■*~ PI Log
■ Ol "".
■ '. .
cef-,
L3 00
0°3 ' -
o
i
O
I—
o o
*~ PI Log
y O'.
iii i r~r prm
rsj
PI Log
<
c
o
o
53
EP 1 561 759 A1
FIG. 11 B
OQ
CD
LU
Q_
CO
^3
OO
CD
CJ3
o
I —
CD
UJ
I —
UJ
Q_
ce:
i- o
CO
o
o
. • -:. •.
3 d
.... •
P 5 ) . ' '
CO*
O
iikc- - :;
• - ■ > -j
/-•<©
com
O
mil i i I |iiiii i i i jiiiu i I
n c-j
o o o
PI Log
- |ui m i i
o
- T-' \ • ' • . * .
' 130
cog-" 1 ' ■
o 00 .
- CD
O
I
O
- o
- o
- o
o
I
O
- o
O
PI Log
o
O
.- .: • > % *
O -: •
- • * ■ \-
- fx . . •
iA . , . V - '
0°
•S.f(N"t^ s i:
0 irv
_ CD
r- CD
CTJ
O
I
o
- o
PI Log
« » T-. . ". . .
O^ - • - -.- •
r o
o
O
o
PI Log
<
c
o
u
<
c
o
o
54
EP 1 561 759 A1
FIG. 12A
PI Log
o ;
a-"-..
■
_ CD
■ O
cn
o
i
O
O
PI Log
<°-i-
.* "*">"*
T— • - * r- - »*
_ o
_ o
o
I
O
- o
PI Log
PI Log
<
c
o
o
<
c
o
o
55
EP 1 561 759 A1
FIG. 12B
CD
csi
H—
<t
eel
LU
Q_
co
UJ
CO
o
o
■■.-x*5 •
<..*-•. • v.-
Q~ , l<ccc/ ' •
1 * I 11 1 1 1 1 I I f imTm-r 1 [ fi ll I I I I
3*
r
o .
CD
CD
o
PI Log
PI Log
o
I —
•<
LU
Q_
CO
LU
CD
CO
CO
PI Log
<«>
r- CD
o
CD '
o
.CD
II I I I jrrm i I I — J'll in I I (111 n i i i
co csi ^ — o
CD O CD O
PI Log
c
o
o
o
o
56
EP 1 561 759 A1
FIG. 13A
VECTOR
10° 10 1 10 2
ANNEX I N V
FIG. 13B
VECTOR
10 3 7
10*
10 1 -
A1
6.8
^72.2£g£ 8.0
A2
13.1
—
10° 10 1 10 2
ANNEX IN V
io 3 1aT
10 2 l
10 1 i
6 7
2D7 DB
A2
75.3
1CNA3 ..
I 5,4
A#3k
12.6.r-
10 2 -
10 1 -
10°-
10° 10 1 10 2 10 3
ANNEX IN V
2D7DB
!ai
:16.4 .
* * * *
A2 .
44.8..-
^ ^^^^^
l23.4§g&
15.3£v^
i > mm i i i .mil \ i i rrtn
10 3
10° 10 1 10 2
ANNEX IN V
10 3
57
EP 1 561 759 A1
FIG. 13C
VECTOR
: 6 8
2-.
10
10 1 -
A2
23.3 - •' • . •
• •: •.. .-; « ' w: ~.-=». : •
2D7 DB
10 j 1a1
13.5 .
r' . i m, , i i n..n i i i-. i i.
10° 10 1 10 2 10 3
ANNEX IN V
ION
A2
50.5-
ANNEX IN V
"j • rrrrj ■, i rrm^ — . , Vmuj — . mrra
10° 10 1 10 2 10 3
FIG. 1 3D
VECTOR
10 3 "|
A1 .
4-8-
A2
3 7 -_> ••
10 z l
*."">>:
• V*. "
•..* *• i
10 1 i
' •"'•/?*. *
10°i
At „-
0.4
' i i imn i : 1 11.111 — I i mil.
2D7 DB
10 ¥C
311.6-
10 2
f :'
10 1
100-4A3^&
332,7;££g£
10° 10 1 10 2 10 3
ANNEX IN V
i mi., i i i rmn J 4 .nul l
10° 10 1 10 2 10 3
ANNEXIN V
58
EP 1 561 759 A1
59
EP 1 561 759 A1
FIG. 15A
FIG. 15B
3
O
!
co r
iunM^i — flum t > — pram i
r
CM ' -r-
Z> " CD . O
PI Log
jnrrrr: v m
o
CD
-
o
,-"
O
O
O
PI Log
o
o
n-*s.v '»-•■. :■ . • .*
<j$^ :
; :0) I
prnrn jiiii 1 1 r r prm pn:m T ■■■
CM O
5 CD CD o
PI Log
CD
O
o
m
o
CD
film -
CD
PI Log
Z3
^Oj-^ ...
pji 1 1 i i — prr
flTTTT
o
■■<■-.
o — 1
<~0
O O
PI Log
PI Log
60
EP 1 561 759 A1
FIG. 15C
FIG. 15D
m
o
o
9"
>
CM f—
o o
CD
o
i
o
<°-,-
nun • i imiiii ' 1
I/O
< . V. . •.. :.r
■unit ■ |IIU'II | jlllllll 1 jllllll 1 1
OJ t— o
3 " O O O
*~PI Log
_ o
o
O
PI Log
PI Log
mrrrf r "p I BT iT T "|i i iui> r |iui<n
eo cnj ■»- o
O O O O
PI Log
CD
r--
irm rn — ptlTm~t — ptirn i i
O
I
O
- o
; no!
I'll 1 1 1 I I
PI Log
61
EP 1 561 759 A1
FIG. 16A
10'
0 ug/ml
1A1 ■ JA2
-17.3% . -112.6%
o
^ 10'- V fvC
10%A33s$£8^4es-
=61.4%j^S8.7%'-:.-
I ' .'lUllj 1 I I HiB] 1 I IT7TTT] 1 i i I llll
10 u
10 1 10 2 10
FITC Log
10'
2.0 ug/ml
|A1
i33.0%
10
o
IA2-"
. 40.4% •
=i 8.1%$^ 8.6%
i 1 1 ihiij "i n mil) — i i.'iuuj — i i trim
10"
10
FITC Log
10 2
10°
FIG. 16B
io4
0 ug/ml
5A1
-11.9%
10
10S
10°J
• iA2
!22.6%
r ~, ■*»r- ■ ••. . -.f ■
10° lb 1 10 2
FITC Log
2.0 ug/ml
-43.5%
1A2
43.0%;--
:8".8%^tv'i4 7%,c'
■ — i 1 1 m i l) — i i i i nn) — i i i niu) — i i i mil
10°
10 1
FITC Log
10 2
10
62
EP 1 561 759 A1
FIG. 17A
10°T?
0 HOUR
12 HOURS
I Tniu^ — i i* i i>nij — i 1 1 mil] — 1. 1 mm
10° 10 1 10 2
FITC Log
10 3
10
10 1 10 2 10 3
FITC Log
24 HOURS
10'
BA1
"66.4%
10 1 -
10°4
>k2
"2.1%
4-
— ;rg. -
i 1 1 iii iii — r
10°
38 HOURS
Q.
10 1 -
FITC Log
FITC Log
63
EP 1 561 759 A1
FIG. 17B
0 HOUR
12 HOURS
;78j^fi&ygo.6%
■ ■'■ r *uunr i i nnq " i rrmn; i i nun
10°
10 1
FITC Log
10 2
10
10*
SA1
-43.5%
"'
o
10 1 -
10°-J
5A2
M.7%
..-•.iV.'- - -"-. I
■ it/ ^ I
54.8%-* 1 0.0%
"r'i di i ii| i < i i iiii) i { uuu\ — t i liiui
10'
10 1 10 2
FITC Log
10
24 HOURS
38 HOURS
i46.9%.< !0.0%
10
10 1 10 2 10 3
10°-J
3A3JL,
:52.5% u
— i i uuaj" i : l iiin) » miii i
FITC Log
10 u
10 1 10 2 10 3
FITC Log
64
EP 1 561 759 A1
FIG. 18A
FIG. 1.8B
CQcsl
mrrrr
■5
co
o
CQCOv
PI Log
PI Log
CD
CM
m
•1/
CQ
tiir
co
CO
CO r^i^LSsj
05
o
1
Jo
1011
OH
ii-
o
PI Log
65
EP 1 561 759 A1
FIG. 19
So
N
+
ml,:
n i i jiiiii
CM
C3
ttt — iram i i
CO.
csi
LL.
-jnn
O
77i V'
Log
3
CD m, .*v
co
G0--
mu i r i — jnrrrrn
'o CD
PI Log
csj :
CO i- 1 ■■ ' .' ~:
m co r^s
frtrtn < 1 1
-o 0
PI Log
PI Log
. .... - ' " c"- ■ 1
_3b
o
Ti m n — [nun i i — I'lii i ■ i i — juuj i
PI Log
66
EP 1 561 759 A1
3
3
C o
>-
o
C_3
+
i
N
+
20
i . -.'l. '
■■ •- - . -
to ■„
m cNi; : 5 . .. .
-.*.■'', -. - "i . E
■ ■ " . " E
ii-ii- "■*;.-,
' ' '
o N
CD
□QCM
lllill l ."1 II 11! 1 1 1 1
•£2oi '■ T
nrm 1 1 i mum i
CD
PI log
o
CD .
3
inn i i ~i — pwiir; i — p
o ~o o
CD
o
ZJ
>-
QQ
<C
a
CM
mcvi
ill'm i' t — [inn > i I
'<=>
•-co co" '
m-q-__
- jmrrrTT
CD
O
PI Log
PI Log
■ "H
' " • '• *- l;'*' '»
' * l. - " 1 i •
^g. -
- 'co
CO
in 1 1 — in i n 1 1 1
mco-' •
— nun i i i
CD
o
o
PI Log
67
EP 1 561 759 A1
68
EP 1 561 759 A1
FIG. 22
200 . 500 1000
2D7DB (ng/ml )
69
EP 1 561 759 A1
FIG. 23
UNTREATED
2D7DB
15 MINUTES
CYTOCHALAS I N D
+
2D7DB
15 MINUTES
70
EP 1 561 759 A1
71
EP 1 561 759 A1
FIG. 25
0 20 40 60 80 100 120 140
DAYS AFTER TUMOR TRANSPLANTATION
VEHICLE — 2D7 DIABODY
72
EP 1 561 759 A1
FIG. 26A
FIG. 26B
• ■
*- -.* '
■ ■ \\ \
10°
10' icr
FITC
File:
Z0021195.LMD
yii Quad % Total
1CT 10*
UL
UR
LL
LR
4.94
20.26
65.33
9.47
o
O.
1 . ,"
!
i
•
■
"J
• V
File:
Z0021198.LMD
Ouad % Total
UL 2.04
UR 22.04
LL 65.98.
LR 9.94
■ ■ ■ . ..X- • .
" -v • 7 J- -J : • :
10'
FITC
p
1
File:
Z0021194.LMD
10"
10 10
FITC
10 10
UL
UR
LL
LR
7.23
45.44
36.01
11.32
m
O
1°'
!0f
i i i fii n'
10" 10'
FITC
,2 a A3 1
File:
Z0021197.LMD
Quad % total
10 10
UL
UR
LL
LR
2.37
30.29
52,65
14.69
2*
O.
■ : —
-
.
i * . -i.
10 u
10" 10 2
FITC
File: ^
Z002fl93.LMD
Quad % Total
UL 6.89
UR 42.35
LL 40.76
LR 1 0.00
10° 10
o.
1
. i.. ■
■ . ^
? >^
File:
Z0021196.LMD
Quad % Total
UL
UR
LL
LR
3.16
33.60
48.20
15.04
10° 10 1 10 2 10 3 10'
FITC
73
EP 1 561 759 A1
FIG. 26C
FIG.26D
CO -
10°
■ rt tht i v
r\ * a
10' 10 2
FITC
File:
Z0021202.LMD
Quad % Total
l W | ' HlH f|
10 3 10'
UL
UR
LL
LR
2.07
31.24
56.25
1 0.44
File:
Z002I205.LMD
Quad
% Total
UL
1.20
UR
28.74
LL
63.74
LR
6.32
10 10"
FITC
■
• ■
"1
!:
■ i . •
■ ' ...
- • • ■
' M^-
■ •.• .
File:
Z0021 201.LMD
Quad % Total
UL 1.98
UR 37.69
LL 30.44
LR 29.89
: ; , • > r/- - > v
FITC
_o.
o •
O-
10 u
Ti ll * ' I
n "1 1
10 10'
FITC
File:
Z0021204.LMD
Quad
% Total
UL
0.91
UR
22.71
LL
40.28
LR
36.10
10 10
o
Q.T-"
10° 10 1
• '-IS'
File:
Z0021
Quad
199.LMD
% Total
10 2 10 3 10 4
UL
UR
LL
LR
2.46
40.37
31.00
26.17
FITC
_o.
o -
:~t ^ i n ' urn
10 10 1 10 2 1
File:
Z0021203.LMD
Quad
% Total
UL
0.93
UR
24.60
LL
42.03
LR
32.44
FITC
(f 10
74
EP 1 561 759 A1
FIG. 26E
o
CO
o
o
o
o.
*
- •
File: Z0021208.LMD
1
Quad
% Total
UL
1.95
!
UR
15.84
LL
LR
77.05
5.16
10° io 1 10 2 1b 3 10
FITC
o
_o
•
■
••
v Z
t iv. ■
File: Z0021207.LMD
Quad
% Total
UL
1.51
UR
55.82
LL
27.05
LR
15.62
FITC
o
o
O.
File: Z0021206.LMD
Quad % Total
'
UL 1 .40
UR 57.54
LL 20.64
LR 20.42
* ■ . <„.
■ . *•■" >
- : -
. *• * * *
10 10 10 10 10
FITC
75
EP 1 561 759 A1
INTERNATIONAL SEARCH REPORT
International application No.
PCT/JP03/13063
A. CLASSIFICATION OF SUBJECT MATTER
Int. CI 7 C07K16/18, C12P21/08, A61K39/395, A61P35/00, A61P37/02,
A61P43/00
According to International Patent Classification (IPC) or to both national classification and IPC
B. FIELDS SEARCHED
Minimum documentation searched (classification system followed by classification symbols)
Int. CI 7 C07K16/18, C12P21/08, A61K39/395, A61P35/00, A61P37/02,
A61P43/00
Documentation searched other than minimum documentation to the extent that such documenls are included in the fields searched
Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
WPI (DIALOG), BIOSIS (DIALOG) , JSTPlusu ( JOIS) ,
GeneBank/EMBL/DDBJ/GeneSeq, Swiss Prot/PIR/GeneSeq
C. DOCUMENTS CONSIDERED TO BE RELEVANT
Category*
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
X
A
GENESTIER L. et al . , Fas-independent apoptosis of
activated T cells induced by antibodies to the HLA
class I al domain.. Blood i997 r Vol.90, No. 9,
pages 3629 to 3639
1
4-
-3
23
X
A
MATSUOKA S. et al . , A novel type of cell death of
lymphocytes induced by monoclonal antibody without
participation of complement., J. Exp .Med . 1995,
Vol.181, No. 6, pages 2007 to 2015
1
4-
-3
23
X
A
FAYEN J. et al . , Negative signaling by anti-HLA
class I antibodies is dependent upon two triggering
events., Int . Immunol . 1998 , Vol.10, No. 9,
pages 1347 to 1358
1
4-
-3
-23
fx - ] Further documents are listed in the continuation of Box C. j^] See patent family annex.
* Special categories of cited documents: "1" later document published after the international filing date or
"A" document defining the general state of the art which is not priority date and not in conflict with the application but cited to
considered to be of particular relevance understand the principle or theory underlying the invention
"E" earlier document but published on or after the international filing "X" document of particular relevance; the claimed invention cannot be
date considered novel or cannot be considered to involve an inventive
"L" document which may throw doubts on priority claim(s) or which is step when the document is taken alone
cited to establish the publication date of another citation or other "Y" document of particular relevance; the claimed invention cannot be
special reason (as specified) considered to involve an inventive step when the document is
"0" document referring to an oral disclosure, use, exhibition or other combined with one or more other such documents, such
means combination being obvious to a person skilled in the art
"P" document published prior to the international filing date but later document member of the same patent family
than the priority date claimed
Date of the actual completion of the international search
05 November, 2003 (05.11.03)
Date of mailing of the international search report
18 November, 2003 (18.11.03)
Name and mailing address of the ISA/
Japanese Patent Office
Facsimile No.
Authorized officer
Telephone No.
Form PCT/tSA/210 (second sheet) (July 1998)
76
EP 1 561 759 A1
INTERNATIONAL SEARCH REPORT
International application No.
PCT/JP03/13063
C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT
Category*
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
Y
A
Y
A
Y
A
ONO K. et al., The humanized anti-HM1.24 antibody
effectively kills multiple myeloma cells by human
effector cell-mediated cytotoxicity., Mol .Immunol.
1999, Vol.36, No. 6, pages 387 to 395
OHTOMO T. et al . , Molecular cloning and charac-
terization of a surface antigen preferentially
overexpressed on multiple myeloma cells., Biochem.
Biophys .Res .Commun. 1999, Vol.258, No. 3, pages 583
to 591
OZAKI S. et al., Humanized anti-HM1.24 antibody
mediates myeloma cell cytotoxicity that is enhanced
by cytokine stimulation of effector cells.,
Blood 1999, Vol.93, No. 11, pages 3922 to 3930
1-4
5-23
1-4
5-23
1-4
5-23
Form PCT/1SA/210 (continuation of second sheet) (July 1998)
77
Live Music Archive
Librivox Free Audio
Metropolitan Museum
Cleveland Museum of Art
Internet Arcade
Console Living Room
Books to Borrow
Open Library
TV News
Understanding 9/11